The Human Microbiota, Infectious Disease, and Global Health

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Perspective

The human microbiota, infectious disease, and global health: challenges and opportunities Abraham J. Waldman, and Emily P Balskus ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00232 • Publication Date (Web): 05 Dec 2017 Downloaded from http://pubs.acs.org on December 6, 2017

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ACS Infectious Diseases

The human microbiota, infectious disease, and global health: challenges and opportunities

Authors: Abraham J. Waldman and Emily P. Balskus*

Author Affiliations Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, MA 02138, United States

Corresponding Author *E-mail: [email protected]

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ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Despite significant advances in treating infectious diseases worldwide, morbidity and mortality associated with pathogen infection remains extraordinarily high and represents a critical scientific and global health challenge. Current strategies to combat these infectious agents include a combination of vaccines, small molecule drugs, increased hygiene standards, and disease-specific interventions. While these approaches have helped to drastically reduce the incidence and number of deaths associated with infection, continued investment in current strategies and the development of novel therapeutic approaches will be required to address these global health threats. Recently, human and vector-associated microbiotas – the assemblages of microorganisms living on and within their hosts – have emerged as a potentially important factor mediating both infection risk and disease progression. These complex microbial communities are involved in intricate and dynamic interactions with both pathogens as well as the innate and adaptive immune systems of their hosts. Here, we discuss recent findings that have illuminated the importance of resident microbiotas in infectious disease, emphasizing opportunities for novel therapeutic intervention and future challenges for the field. Our discussion will focus on four major global health threats – tuberculosis, malaria, HIV and enteric/diarrheal diseases. We hope this Perspective will highlight the many opportunities for chemists and chemical biologists in this field, as well as inspire efforts to elucidate the mechanisms underlying established disease correlations, identify novel microbiota-based risk factors, and develop new therapeutic interventions.

KEYWORDS: microbiota, infectious disease, global health, tuberculosis, malaria, HIV, enteric disease


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Main text:

Worldwide morbidity and mortality caused by infectious agents is one of the most substantial scientific challenges and global health threats of our time. Beginning in the early 20th century, the discovery and development of anti-infective small molecule therapies, coupled with improvements in sanitation and personal hygiene, resulted in a drastic reduction in deaths and disease caused by infections. However, these advances were primarily realized in industrialized, high-income nations. While low- and middle-income countries have also benefited from these achievements, the global disease burden from infectious agents remains staggeringly high in many parts of the world, including Africa, India, and South East Asia. Collectively, infectious agents result in approximately 9 million deaths annually as well as significant economic costs.1 Currently, strategies for prevention and treatment of these diseases encompass a wide range of approaches including vaccines, small molecule drugs, increased hygiene practices, as well as disease-specific interventions e.g. condom usage to prevent HIV transmission. While these interventions have saved millions of lives, the unacceptably high death rate combined with growing resistance to anti-infectives necessitates sustained investment in novel strategies to combat these pathogens.

In this Perspective, we discuss the growing evidence that both vector- and humanassociated microbiotas play an important role in infectious diseases that threaten global health.2–4 The human microbiota refers to the trillions of microorganisms (bacteria, archaea, viruses and microscopic eukaryotes) that inhabit the skin, respiratory tract, urogenital tract, gastrointestinal tract, and other body sites.5 These complex microbial communities play integral roles in both healthy physiology as well as various disease states.6 Although the definition of a healthy, ‘core’



microbiota at any body site has remained elusive, changes in the diversity and composition of these communities (dysbiosis) are associated with various metabolic, cardiovascular, gastrointestinal, autoimmune, and infectious diseases.7 These links have largely resulted from studies that have used DNA sequencing to profile the composition and gene content of patient microbiomes, as well as experiments involving transplantation of microbes into germ-free model hosts (gnotobiotics). While these descriptive studies have provided valuable insights, we still largely lack a mechanistic understanding of the roles these microbial communities play in host biology. Research into the human microbiota’s role in health and disease is at a pivotal and exciting time as we move from asking observational questions (‘who is there?’), to deciphering the biological functions of these organisms (‘what are they doing?’). The dramatic success of fecal microbiota transplants (FMT) in treating recurrent Clostridium difficile infections is also fueling growing interest in microbiota-based therapies.8,9

We believe that a molecular understanding of these microbial communities will inform novel therapeutic intervention strategies to combat the global health problem of infectious diseases, and that chemists and chemical biologists are poised to contribute to this important objective. Here, we discuss the roles that both human- and vector-associated microbiotas may play in four infectious diseases of major global health concern: (1) tuberculosis, (2) malaria (3) HIV, and (4) enteric and diarrheal diseases (Figure 1). We highlight key findings that have revealed exciting opportunities for microbiota-based solutions to disease prevention, as well as the challenges faced in implementing these approaches. Our hope is that this Perspective will inspire further research in this area.


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Figure 1. Overview of the human and vector microbiotas as well as associated infectious diseases discussed in this Perspective.

Strategies for microbiota-based interventions While the mechanistic underpinnings of the microbiota-disease etiologies discussed in this Perspective differ, one can envision applying related microbiota-based intervention strategies. Here, we provide an overview of general methods for microbiota manipulation that may be applicable for preventing or treating a variety of infectious diseases, as well as the advantages and challenges associated with their application (Figure 2). Altering community structure and function in these complex microbial ecosystems with precision, stability, and reproducibility is a formidable problem. However, studies in animals and humans are revealing important insights that will inform future development in this area.



Prebiotics/Diet. Diet can have a significant effect on gut microbiota composition by providing nutrients that promote the growth of different types of bacteria. Prebiotics are classes of dietary compounds, including fructooligosaccharides, bran, and inulin, that stimulate the growth of beneficial commensal bacteria, most commonly Lactobacillus and Bifidobacterium (Figure 2A).10 Prebiotics are easy to administer and have reproducibly demonstrated an effect on the overall composition of the gut microbiota.11 However, they function in a relatively non-specific way by promoting the growth of multiple species and may not provide a long-term, stable adjustment of the community. A better understanding of the molecular mechanisms by which different dietary compounds and their metabolic end products stimulate microbial growth may enable a more rational use of prebiotics.

Probiotics. Probiotics are specific microbial species, defined consortia, or complex communities that are administered to replenish or introduce beneficial functions into a host microbiota (Figure 2B).12 Probiotics are generally ingested with food products, but they can also be administered via oral encapsulations, and through FMT. The striking success of FMT in treating recurrent C. difficile infections has provided perhaps the strongest proof of concept for gut microbiota manipulation as a therapeutic strategy.13 Probiotic strains have also been engineered to participate in new interactions with the host and/or gut microbiota, including carbohydrate metabolism and the local delivery of IL-10 to treat inflammatory bowel disease (IBD).14,15 These engineered systems offer the opportunity to introduce functions into the gut that would not be available using naturally-occurring microbial species. Probiotics – both natural and engineered – have the advantage of directly introducing specific biological functions into communities, however it is challenging for these organisms to colonize an established, complex microbiota.16


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We also lack a mechanistic understanding of how these species interact with the host and other gut microbes. Furthermore, multiple scientific and regulatory issues currently surround the introduction of genetically engineered organisms given the challenges of biocontainment and horizontal gene transfer.

Synbiotics. Synbiotic interventions combine the introduction of new microbe(s) into a community with co-administration of a substrate to support growth of the new strain(s), thereby taking advantage of both prebiotic and probiotic strategies.17 While synbiotics may lead to more efficient and stable colonization of probiotic strains, an understanding of the specific nutritional requirements of the microorganism being introduced is required.18,19

Antibiotics. The use of current broad-spectrum antibiotics has a profound and long-lasting effect on gut microbiota composition.20 However, such large-scale manipulation of community structure is often not desired as it can increase susceptibility to infectious disease and promote the spread of antibiotic resistance.21 The development of narrow-spectrum antibiotics, which kill a smaller, defined set of target bacteria, would provide a more precise set of tools for community manipulation (Figure 2C). This strategy has demonstrated significant promise recently with the development of fidaxomicin (Dificid) a macrocyclic lactone natural product that selectively eradicates C. difficile. While this strategy may limit collateral damage to beneficial microbes, it will only be effective when deleterious activities are confined to single species or closely related organisms. Small molecule antibiotic candidates that were abandoned because of their narrow spectrum could find novel repurposing in this context.



Phage. Phages are viruses that target bacteria and are typically extremely specific for their target organism (Figure 2C). In natural ecosystems, phages are highly abundant and involved in numerous important process that influence community dynamics and functions. They manipulate species abundance and diversity through selective killing and introduce beneficial new traits such as carbohydrate metabolism, toxin production, and antibiotic resistance through gene transfer.22 Phage-based interventions currently under investigation include clinical trials assessing the efficacy of phage therapy to treat Escherichia coli and Pseudomonas aeruginosa infections in burn wounds (PHAGOBURN) and the identification of phages from human stool capable of preventing colonization of multi-drug resistant Enterobacteria (PHAGO-BMR).23,24 Phage therapy allows for species-level precision in manipulating the microbiota without toxicity to human cells. However, this strategy requires identifying phage that will infect bacteria of interest and may not be as effective for targeting multiple organisms or widely distributed functions. Increased understanding of the roles phage play in the human microbiota will not only uncover additional phages, but may also reveal new ways to use this therapeutic approach.

Small Molecule Inhibitors. When specific microbial metabolic activities have been linked to disease, it may be advantageous to inhibit these pathways directly, especially if the function of interest resides in phylogenetically diverse and/or in otherwise beneficial organisms (Figure 2D).25 The recent development of small molecules that inhibit bacterial β-glucuronidase enzymes illustrates the potential of this approach. These enzymes remove the sugar from glucuronide drug-conjugates generated by host metabolism, reactivating therapeutics in the gut lumen and causing unwanted side effects. β-glucuronidase inhibitors that were selective for gut bacterial versus host enzymes prevented the toxicity of the anticancer drug irinotecan in mice, providing


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critical proof-of-concept for this intervention strategy.26 Small molecules that inhibit specific microbial functions could provide exquisite precision in manipulating communities, offering temporal and reversible control over the inactivation of individual activities. They may also be well suited to address the challenge of inter-individual variation in microbiota composition. In contrast to many of the approaches highlighted here, the pathways for clinical development and translational application of small molecules are well defined. However, development of such inhibitors may necessitate a detailed, mechanistic knowledge of the underlying disease etiology that is often lacking.



Figure 2. General methods (left panel) for the manipulation of microbiota composition and functions with specific examples included (right panel). A) Prebiotics can support the preferential growth of beneficial organisms with communities. B) Probiotic strategies involve introducing single strains or consortia of either natural and engineered microorganisms into a community. C) Both broad- and narrow-spectrum antibiotics as well as phage can remove microbes from communities. D) Non-lethal, small molecule inhibitors can selectively inhibit specific microbial functions within complex communities.


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Tuberculosis

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis complex, remains one of the most devastating global health threats. In 2015, approximately 10.4 million people became ill with TB and 1.8 million individuals died.27 Failure to comply with strict antibiotic regimens results in incomplete sterilization of the infection and generation of multidrug-resistant (MDR) strains that no longer respond to first-line therapies. As a result, an estimated 500,000 individuals developed MDR-TB in 2015.27

The M. tuberculosis life cycle during TB infection is a complex series of interactions with the human host that are not fully understood.28,29 Infection begins when aerosolized M. tuberculosis is inhaled into the lungs and the responses of both the innate and adaptive immune system fail to completely eradicate the infection. Following this initial event, M. tuberculosis exists in a state of ‘immunological equilibrium’ known as latent TB infection (LTBI) where it resides primarily within alveolar macrophages. At this stage its growth is heavily attenuated, but adaptive mechanisms prevent its complete destruction. In approximately 5–15% of infected individuals, unknown factors disrupt this equilibrium, resulting in reactivation and growth of M. tuberculosis with progression to active TB illness and eventually death. A more complete mechanistic understanding of why our immune response fails to eradicate the initial infection and what factors allow for later reactivation could help in developing new therapeutic intervention strategies to prevent or fight TB infection and disease progression. Given our rapidly growing understanding of gut microbiota-immune system interactions and our more



recent appreciation of the importance of the lung microbiota for healthy respiratory immunology, it seems likely that these communities affect the immune response to TB infection and reactivation.2,30

The microbiota of the lower respiratory tract (lung) is significantly understudied compared to other body sites. This discrepancy reflects the challenges of sampling and analyzing this community, including extremely low bacterial density, contamination from the upper respiratory tract (nasopharynx and oropharynx) during sampling, and the lack of non-invasive sampling methods. Despite these obstacles, culture-independent sequencing-based studies have begun to provide insights into the dominant phyla and genera of the ‘healthy’ lung microbiota. As with more extensively studied body sites, considerable inter-individual variation exists in this habitat.31,32 Due to a combination of nutritional, physiochemical and immunological factors, the lung is considered to be a largely inhospitable environment for bacterial growth. Given the similarity between microorganisms found in the lung and the URT, it is generally believed that the lung microbiota represents a group of transient organisms largely determined by immigration from the URT and elimination.2,33,34 Despite its transience, it is well-accepted that the lung microbiota is important in the development of the respiratory tract, for maintenance of proper immune homeostasis, and in colonization resistance, although knowledge of its specific functional roles in these processes is incomplete.

Efforts to characterize the relationship between TB infection and the composition of the lung microbiota have not yet provided a clear consensus as to whether community composition is correlated with disease.30,33 In contrast, dozens of studies profiling the lung microbiota have


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shown significant correlations between changes in community composition and other diseases, with many conditions associated with an increase in bacterial diversity.34 The lack of a consensus emerging from investigations in TB infection may be a result of the small sample sizes used as well as differences in sampling techniques and the general difficulties associated with sampling the lung microbiota. Future efforts should include larger sample sizes and standardized sampling methods to accurately compare healthy subjects with infected patients.

Although no clear relationship has emerged between the lung microbiota and TB infection, this community has been implicated in modulation of mucosal immunity in the respiratory tract, which is expected to be an important factor in infection. In healthy human subjects, dominant genera in the lung, such as Prevotella and Veillonella, have been associated with changes in immune system physiology, including increases in pro-inflammatory lymphocytes and cytokines and an attenuated TLR4 response in alveolar macrophages.35 Additionally, similarly to the gut microbiota, members of the lung microbiota have been proposed to induce antimicrobial peptide production by the innate immune system.33

Studies examining the influence of the gut microbiota on the respiratory immune system via the ‘lung-gut axis’ have suggested these gut organisms may influence both risk of TB infection and the development of active TB.36 In a study with cynologous macaques, individuals were less likely to develop active TB when challenged with M. tuberculosis if they were also infected with Helicobacter pylori.37 In contrast, when mice carry the commensal bacterium Helicobacter hepaticus they are more susceptible to TB infection.38 While gut microbes correlate with infection risk and disease progression in these animal models, understanding the molecular



bases for these effects, and assessing whether similar correlations exist in humans, are important future challenges.

Recently, an association was reported between the production of serum and pulmonary short chain fatty acids (SCFAs), an increased abundance of anaerobes in the lung (including Prevotella, Veillonella, and Haemophilus), and a heightened risk of TB infection in humans (Figure 3).39 The mechanism underlying this association is hypothesized to be the inhibitory effect of butyrate on the induction of the pro-inflammatory cytokines IFN-γ and IL-17A, which are normally induced by M. tuberculosis antigens. This intriguing finding represents a rare example of a potential mechanistic link between the lung microbiota and susceptibility to TB infection. Moving forward, it will be important to explore this proposal in animal models of infection to determine whether a causal relationship exists between SCFA production and increased risk of TB.

Overall, our current knowledge of the roles the lung and gut microbiota play in TB infection and disease progression is very limited, though preliminary studies suggest that interactions between these microbial communities and the host immune system affect both the initial infection and activation of latent TB. Additional studies in both humans and animal models will be necessary to identify the specific functions of lung and gut microbes that mediate these effects. Given the possible connection between SCFAs and increased TB risk, a potential preventative intervention could involve reducing the production of these microbial fermentation products either through elimination of SCFA-producing anaerobes from the lung, redirecting this


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metabolism via prebiotics, or through the specific inhibition of SCFA-producing metabolic pathways in this environment.

Figure 3. Lung microbiota composition is correlated with heightened TB infection risk. A) Increased anaerobe abundance in the human lung is associated with increased butyrate production, lower IFN-γ, and decreased IL-17A levels, which may heighten risk of TB infection. B) Butyrate production by anaerobic bacteria may inhibit the production of protective, proinflammatory cytokines IFN-γ and IL-17A by CD4+ and CD8+ T cells.



Malaria

According to WHO estimates, there were approximately 212 million cases of malaria and 429,000 deaths resulting from the infection in 2015.40 Children under the age of 5 are particularly susceptible to this disease, accounting for 70% of malaria deaths. Encouragingly, between 2010 and 2015 new infection rates fell by 21% worldwide and global mortality rates fell by 29%. But despite these advances, malaria remains a major public health threat. New measures are need to combat the disease, especially considering the growing threat of artemisinin-resistant parasites.

Malaria infection occurs when female Anopheles mosquitoes (vectors) harboring humanassociated Plasmodium parasites, most commonly Plasmodium falciparum or P. vivax, bite humans to obtain a blood meal.41 The Plasmodium parasite has a complex and intricate life cycle that involves several distinct stages in both the human and mosquito.42 Successful progression of parasites through these various stages is required for successful transmission of the disease. Currently, the most effective strategies for addressing malaria are prophylactic interventions that focus on preventing transmission by stopping mosquito bites, including use of insecticide-treated mosquito nets and insecticide sprays indoors. Additional prophylactic measures involve administration of monthly courses of several drugs prior to infection, which is particularly important for children under 5 years of age. With prompt diagnosis, malaria infections can be cured with artemisinin combination therapy (ACT), although the rise in artemisinin-resistant parasites threatens the effectiveness of this treatment.43


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While research on malaria prevention and treatment has focused primarily on vector control and novel small molecule therapies to prevent or cure infection, a growing body of work has suggested that the human and mosquito microbiotas may have an important role in both infection risk and disease progression. These studies have not only provided insights into potential strategies to combat malaria, but have also enabled exciting proof-of-principle studies that await further translational exploration.

Some of the earliest research investigating the relationship between the human microbiota and malaria infection was motivated by the well-established observation that Anopheles gambiae mosquitoes are attracted by human-specific odors. It was first proposed in the late 1990s that these odors may be produced by human skin bacteria, as mosquitos were only attracted to human sweat after it had been incubated for one to two days.44 The human skin is home to a complex consortium of microorganisms that displays low diversity at the phylum level but high diversity at the species level.45 While there is considerable variability between the skin microbes present at the same body site in different individuals, as well as between the communities from different sites within the same individual, community structure at these various locations is generally consistent over time. Changes in the composition of the skin microbiota have been associated with various conditions, including acne and atopic dermatitis.46

Given the inter-individual variability of the skin microbiota and the established ability of skin-associated bacterial species to produce volatile small molecules, it was postulated that humans may attract Anopheles mosquitoes to different degrees depending on the profile of volatiles produced by this microbial community. Subsequent investigations have identified



specific strains of skin bacteria including Staphylococcus epidermidis, Corynebacterium minutissimum, and Bacillus subtilis that produce a panel of aliphatic, oxidized small molecules, including alcohols, aldehydes, ketones, esters, carboxylic acids and sulfides, which appear to attract mosquitoes (Figure 4).47,48 Additionally, the diversity and abundance of the skin microbiota on the feet has a significant association with the attractiveness of individuals to mosquitoes, with an increased abundance of Staphylococcus spp. associated with increased attractiveness and increased Pseudomonas

spp. abundance correlated with lowered

attractiveness.49 Further studies will be required to determine whether these volatiles are produced directly by bacteria on human skin in high enough concentrations to mediate attraction and how their relative abundances in these complex mixtures affects attractiveness. Identifying a minimal set of attractive volatiles and bacterial species will be helpful for informing intervention strategies.

Figure 4. Human skin bacteria produce volatile compounds that are attractive to A. gambiae mosquitoes. Mosquitoes may use these compounds as chemical cues to seek their human host


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when searching for a blood meal. These compounds include isovaleraldehyde (1); 2,3butanedione (2); dimethyl disulfide (3); isovaleric acid (4); butyl isobutyrate (5); and n-butanol (6).

Investigations into the skin microbiota’s role in malaria infection highlight several opportunities for intervention. Combining attractive compounds derived from the skin microbiota together with insecticides in traps could serve to both attract and kill mosquitoes when strategically placed away from human activity. Additionally, efforts could be made to remodel the skin microbiota by eradicating or replacing bacteria that produce attractive chemicals, to shift metabolism away from volatile production using probiotics, or to inhibit the production of these volatile compounds with small molecules that target specific bacterial enzymes involved in their biosynthesis.

Considering the complex lifestyle of Plasmodium parasites, it is perhaps not surprising that these organisms appear to be affected by the microorganisms present at multiple human body sites. In healthy humans, anti-α-gal antibodies produced against the α-gal sugar present in the cell walls of certain commensal bacteria were shown to also target the Plasmodium parasite, and higher levels of these circulating antibodies are associated with decreased risk of infection.50 Additionally, higher proportions of Bifidobacterium and Streptococcus in the human gut microbiota are correlated with a lowered risk of malaria infection, but no association was found between gut community composition and the later development of severe malaria.51 While these studies present intriguing associations, a recent study in mice demonstrated strong causal evidence for the role of the gut microbiota in modulating the severity of the disease.52 Mice with



gut microbiotas containing higher abundances of Lactobacillus and Bifidobacterium had significantly lower parasite burden and mortality after infection with multiple mouse-associated Plasmodium species. Moreover, when antibiotic-treated mice were given laboratory-cultured yogurt containing Lactobacillus and Bifidobacterium species before and following infection with P. yoelli, a drastic reduction in parasite burden was observed. Mice given this intervention had higher titers of Plasmodium specific IgG antibodies following infection, suggesting that changes in the gut microbiota may affect disease progression through modulation of the immune system.

These studies have begun to demonstrate the impact of human gut microbiota on both Plasmodium infection and the development of severe malaria. While mechanistic insight into the protective roles of gut bacteria in malaria is severely lacking, current evidence points towards interactions with the host immune system as a primary driver.50,52 This work points toward manipulation of the gut microbiota as a potential strategy for malaria prevention. Intriguingly, these studies have implicated bacterial species (Bifidobacteria and Lactobacillus) that are already widely used as probiotics. It has also been shown that supplementing the human diet with prebiotics, such as inulin and lactulose, can increase the abundance of these species.53 Studies examining the protective effects of these strategies in humans are needed to help clarify whether their role in malaria protection will extend to real-world settings.

Several distinct stages in the Plasmodium parasite’s life cycle take place in the mosquito vector, raising the possibility that the mosquito gut microbiota may affect parasite survival. Indeed, the development of the parasite in the mosquito gut represents one of the strictest bottlenecks in its life cycle. At this stage, a number of host and microbiota factors are thought to


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heavily restrict the conversion of gametocytes into oocysts, a critical step in the Plasmodium development.54 Associations between mosquito gut community composition and susceptibility to Plasmodium infection have been demonstrated in wild-caught mosquitos, with increased abundance of Enterobacteriaceae correlating with infection.55 Studies using germ-free and conventional mosquitoes have suggested that the gut microbiota protects the mosquito from Plasmodium infection by activating its innate immune system, which in turn non-selectively targets both Plasmodium parasites and gut bacteria.56,57 Additionally, a strain of Enterobacter spp. isolated from a wild-caught mosquito drastically reduced the survival and development of Plasmodium parasites in the mosquito gut with a mechanism of action that appears to involve the direct killing of parasites through generation of reactive oxygen species.58

With evidence that the mosquito gut microbiota can significantly affect Plasmodium infection, recent exciting proof-of-concept studies have explored the feasibility of using genetically engineered mosquito gut commensals expressing anti-Plasmodium factors to kill the parasite.59,60 Researchers engineered two mosquito gut bacterial commensals to produce a combination of anti-Plasmodium peptides and enzymes derived from both natural and synthetic sources. These toxins act through diverse mechanisms, but ultimately block parasite survival and development by either preventing transversal of the parasite across the gut epithelium or through lytic mechanisms.59 Introduction of these engineered, toxin-producing bacteria into mosquitoes via sugar meals drastically reduced parasite burden in the gut following a P. falciparum-infected blood meal. Importantly, one of these engineered strains (AS1) is transmitted rapidly and stably throughout laboratory mosquito populations.60 A major future challenge for this intervention strategy will be demonstrating that the AS1 strain can propagate in wild populations of



mosquitoes and assessing whether the drastic reductions in parasite burden in Anopheles mosquitos will result in decreased malaria infections in humans.

In summary, malaria is a complex and dynamic infectious disease, with numerous factors in both the human host and mosquito vector affecting the survival and development of Plasmodium spp. Over the last two decades, it has become clear that microbial communities in both humans and mosquitos play an important role in the parasite life cycle. These new insights have pointed toward specific strategies for therapeutic intervention that are poised to help combat this global epidemic.

HIV

Since the outbreak of the HIV epidemic in 1980s approximately 35 million people have died from AIDS-related causes. In 2016, it was estimated that approximately 36.7 million individuals were living with HIV, with 1.8 million new infections and 1.0 million people dying from immune deficiency-related complications.61 Unlike tuberculosis and malaria, there is currently no cure for HIV infection. However, lifelong administration of combination antiretroviral therapy is highly effective in blocking viral replication and can suppress viral load enough to dramatically extend life expectancy and prevent transmission of the virus.62 Approximately 54% of adults and 43% of children infected with HIV are currently receiving antiretroviral medicines.61 Because no cure for HIV exists, public health strategies have emphasized early diagnosis and preventative measures including HIV testing, condom usage, and prophylactic use of antiretrovirals.


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HIV infection through heterosexual sexual intercourse occurs at epithelial and mucosal surfaces of the vagina and penis, where HIV primarily targets CD4+ T cells of the immune system.62 Progressive loss of CD4+ T cells, as well as other detrimental effects on the immune system, leads to clinical manifestation of AIDS, with mortality often resulting from comorbidities, including secondary infections and cancer. Bacterial communities at mucosal and epithelial surfaces are important factors that modulate local immunity.3 Given that HIV infects cells of the immune system, it is perhaps unsurprising that the resident microbiota of both the penis and vagina appear to influence HIV infection risk.

A number of large scale epidemiological studies have demonstrated that male circumcision reduces the rate of HIV transmission by approximately 60%, although the mechanisms underlying this phenomenon are unknown.63 Additional work has revealed a drastic change in the penile microbiota post circumcision, offering potential insights into the factors affecting this change in transmission rate. Both the diversity and abundance of bacteria are reduced after circumcision, with the number of strictly anaerobic genera decreasing and the proportion of facultative anaerobes increasing.64,65 It has also been demonstrated that uncircumcised men possess a more pro-inflammatory physiology of the coronal sulcus of the penis including increased production of cytokines and higher densities of CD4+ T cells.66,67

A recent study revealed a potential connection between HIV acquisition and the heightened inflammatory state and higher abundance of anaerobic penile bacteria observed in uncircumcised men (Figure 5A).68 Increased levels of certain anaerobic bacteria, including



Prevotella, were associated with an increased risk of contracting HIV. A 10-fold increase in the abundance of different anaerobic genera correlated to a 54 – 63% increased risk of infection. Moreover, this shift in the microbiota was associated with increased cytokine production, including interleukin-8 (IL-8). IL-8 can lead to the recruitment of HIV-susceptible cells including Th17 helper T cells, providing a mechanistic link between this change in penile microbiota composition and increased susceptibility to HIV infection.

Figure 5. Connections between the penile and vaginal microbiotas and HIV infection risk. A) Higher abundance of anaerobic bacteria in the coronal sulcus of uncircumcised men is linked to a heightened inflammatory state and the recruitment of HIV-susceptible cells, potentially increasing infection risk. B) High diversity, non-Lactobacillus-dominated vaginal microbiotas are associated with increased inflammation and abundance of HIV-susceptible cells, possibly explaining a higher incidence of HIV infection. C) G. vaginalis and other BV-associated bacteria


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degrade the prophylactic drug tenofovir in vitro and are associated with decreased efficacy of this preventative treatment.

Dysbiosis of the vaginal microbiota has been associated with increased inflammation and an enhanced risk for several sexually and non-sexually transmitted infections.69,4 However, defining exactly what constitutes dysbiosis in this community has been challenging. Efforts to characterize the composition of the vaginal microbiota have shown that the vaginal microbiotas of healthy, asymptomatic women are often dominated by a single species of Lactobacillus, including L. iners, L, crispatus, L. gasseri, or L. jensenii.70 In contrast, 20 – 30% of healthy, asymptomatic women have more diverse communities that are not dominated by Lactobacillus, yet appear to retain conserved functions that may be necessary for healthy physiology such as lactic acid production.4 Disruption of this healthy, low diversity community structure may lead to bacterial vaginosis (BV), which is described as a dysbiotic, polybacterial infection marked by increased diversity and a higher abundance of facultative and strict anaerobes.69 This strong association between low bacterial diversity and healthy vaginal physiology is in stark contrast to other body sites, where lower diversity is often correlated with disease, highlighting that community function rather than composition may be the critical factor influencing health and disease.

Recent efforts to understand the role of the vaginal microbiota in HIV infection has suggested that this community is an important risk factor (Figure 5B). Increased levels of proinflammatory cytokines in cervicovaginal lavage fluid has been associated with a heightened risk for HIV infection, consistent with the recruitment of HIV-susceptible immune cells to the



mucosal surface during inflammation.71 A pro-inflammatory vaginal state, marked by increased cytokine production, has been strongly associated with a highly diverse, non-Lactobacillusdominated vaginal microbiota in a study examining otherwise healthy, asymptomatic young women from South Africa.72 Transcriptional profiling suggested that this increased inflammation arises from immune system sensing of Gram-negative bacteria through TLR4, thus promoting lymphocyte recruitment. Further analysis of this cohort revealed that women with vaginal microbiotas containing diverse groups of anaerobic bacteria other than Gardnerella had a 4-fold higher risk of acquiring HIV compared with women whose vaginal microbiotas were dominated by L. crispatus.73 Cervicovaginal lavage fluid from the women with diverse, non-Lactobacillusdominated microbiotas had higher abundances of activated mucosal CD4+ T cells, providing a mechanistic link between vaginal microbiota composition and HIV infection. Strong causal evidence for the vaginal microbiota’s role in promoting inflammation came from subsequent mouse experiments. Germ-free mice inoculated with the vaginal anaerobe Prevotella bivia showed significantly higher levels of CD4+ T cells in the vaginal mucosa compared with mice inoculated with L. crispatus. In combination, these studies demonstrate a role for the vaginal microbiota in mediating risk for HIV infection that parallels that proposed for the penile microbiota, with certain community members promoting inflammation, which results in increased levels of HIV-susceptible target cells at the site of infection.

In addition, recent work has revealed that interactions between the vaginal microbiota and certain HIV preventative therapies may influence transmission rates in women. The CAPRISA 004 study demonstrated that high adherence (>80%) to prophylactic administration of a vaginally-applied, antiretroviral gel containing 1% tenofovir reduced HIV infection rates in


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women by 54%.74 Interestingly, further analysis of this data revealed a significant difference in risk reduction that correlated with the composition of the vaginal microbiota, with less protection observed in women with non-Lactobacillus dominated communities.75 This difference may result from the ability of Gardnerella vaginalis and other BV-associated bacteria common in nonLactobacillus-dominated microbiotas to metabolize tenofovir (Figure 5C). Several of these strains degraded tenofovir in vitro, cleaving its oxy-methylphosphonic acid side-chain to release adenine via an undefined mechanism. Moreover, levels of tenofovir in cervicovaginal lavage fluid were reduced and the drug was detected at a lower rate in women with non-Lactobacillusdominated vaginal microbiotas compared with Lactobacillus-dominated communities. Further studies are needed to determine if G. vaginalis and other bacteria can degrade tenofovir in vivo, to identify the genes and enzymes responsible for this activity, and to assess whether additional factors contribute to the differences in efficacy.

One microbiota-focused strategy for preventing HIV infection that has shown initial proof-of-concept results is genetically engineering probiotic strains to produce anti-HIV effector proteins. These bacteria could potentially be administered orally or delivered into vaginal microbiotas.76 Various strains of Lactobacillus (L. jensenii, L. casei, L. reuteri) and E. coli have been engineered to express several different anti-HIV proteins, including effectors that interfere with target cell binding and membrane fusion.77-81 Some of these genetically engineered strains showed efficacy in reducing HIV infection in in vitro cell culture models. While these initial results are promising, it will be important to translate these results into animal models and address the challenges associated with introducing new strains into these microbial communities.



Investigations into the effects of the penile and vaginal microbiotas on HIV infection have exposed promising opportunities for therapeutic intervention. In men, efforts to improve hygiene standards, including the use of antibacterial soaps to eliminate anaerobic bacteria, could potentially reverse the inflammation associated with these species. Additional strategies that shift the microbiota away from risk-associated genera could also be helpful. Moreover, this work provides further evidence in support for circumcision as a strategy for preventing HIV acquisition in high-risk populations. In women, this work suggests that transitioning the vaginal microbiota toward a Lactobacillus crispatus-dominated community using probiotic- and/or prebiotic-based interventions may be a particularly promising approach for reducing HIV risk.76 Finally, the potential role of G. vaginalis in tenofovir degradation should prompt additional efforts to uncover microbiota-drug interactions beyond the gut microbiota.82 Understanding such interactions could allow for screening of patient samples to inform treatment decisions and guide the development of strategies to prevent drug metabolism, including inhibitors targeting bacterial enzymes.

Enteric and Diarrheal Diseases

Enteric infection and diarrhea is currently the second leading cause of death in children under 5 years of age, accounting for approximately 525,000 deaths annually.83 Major contributors to diarrheal diseases include rotavirus, enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), Vibrio cholerae, and Salmonella enterica Typhi.84 In addition to causing death, chronic exposure to enteric pathogens results in a widespread, yet poorly understood subclinical condition of prolonged intestinal


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inflammation termed ‘environmental enteric dysfunction’ (EED).85 This condition is marked by reduction in epithelial barrier integrity and a decrease in nutrient absorption capacity that leads to chronic malnutrition, stunting, and prolonged morbidity and deficits throughout life. Current efforts to manage this global health crisis include rehydration therapies with oral salt solutions, access to clean drinking water, improved sanitation and personal hygiene, exclusive breastfeeding during first six months of life, and rotavirus vaccination.86 While these strategies have resulted in significant decreases in mortality over the last decade, the death toll remains staggeringly high and morbidities associated with EED have persisted.85 The enormous amount of research on the gut microbiota and its involvement in enteric infection offers novel opportunities to combat these diseases.

Resident microbes affect the ability of enteric pathogens to colonize the human gut and cause disease. The gut microbiota mediate colonization resistance through various direct and indirect mechanisms, including modulating the immune system and its response to pathogens, as well as directly influencing the growth and virulence of these organisms (Figure 6). While the idea of altering the gut microbiota to increase colonization resistance and prevent infection is well established, we are only beginning to uncover the mechanisms behind this phenomenon.3,87 This knowledge may inform interventions to protect against pathogen invasion and disease. Such strategies are expected to help not only in combating acute infections that lead to death, but also in addressing the deficiencies associated with EED by reducing inflammation and promoting expansion of beneficial commensals, which are required for proper nutrient absorption and gut epithelial integrity. Importantly, these microbiota-based strategies are likely to be most effective when administered prophylactically to the populations most often affected by these diseases.



Figure 6. Commensal microbes influence the growth and virulence of pathogens. A) Commensals can occupy metabolic niches by consuming carbon sources that are preferentially used by pathogens. B) Commensal bacteria can inhibit pathogen growth directly by producing inhibitory molecules. C) Commensals can inhibit the production of virulence factors by pathogens.

The prospect of preventing pathogen colonization by administering defined gut microbiota communities and single species has demonstrated significant promise in mouse models of C. difficile, vancomycin-resistance Enterococcus (VRE), EPEC, EHEC, and S. enterica Typhi infection.88-91 This strategy is also being explored in humans, with several


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microbiota-based therapeutics for the prevention of recurrent C. difficile infections currently entering or in clinical trials.92 In addition, a synbiotic approach involving administration of a probiotic strain of Lactobacillus plantarum together with a prebiotic fructooligosaccharide to rural Indian newborns led to a significant reduction of death by sepsis.93 Sepsis is often caused from intestinal pathogens breaching the gut epithelial barrier and entering the blood stream, suggesting that this intervention may be preserving the integrity of the gut epithelial barrier via physical mechanisms (e.g. mucus production), eliciting a host immune system response (e.g. antimicrobial peptide secretion), and/or adherence of L. plantarum to the gut epithelium thereby blocking translocation of pathogens.

Together, these recent studies have demonstrated the

potential of manipulating the human gut microbiota to combat enteric pathogens. Below we describe several mechanisms by which the gut microbiota interacts directly with pathogens to mediate colonization resistance, highlighting the additional opportunities for therapeutic intervention.

The human gut is a highly competitive environment in which microbes vie for diet- and host-derived nutrients. Closely related bacteria often compete for similar niches, as demonstrated by the reduced capability of a specific bacterial strain to colonize the mouse gut when similar strains are already present.8 A nutritional basis for this colonization resistance has been demonstrated in a mouse model involving commensal strains of E. coli and a pathogenic STEC strain.94 Administering to streptomycin-treated mice two strains of commensal E. coli that together consume the five sugars necessary for STEC growth rendered the STEC pathogen incapable of colonization. This finding suggests several strategies for preventing enteric infections, including prophylactic administration of bacterial species that can compete for



nutrients required by pathogens and the use of prebiotics to promote the expansion of commensals that can occupy these key nutritional niches. While this strategy has shown efficacy in simplified animal models, it will be important to determine if these findings translate to more complex gut communities where networks of metabolic interactions provide access to a wider range of nutrients. Indeed, pathogens can use nutrients and carbon sources not commonly consumed by commensals.95 Implementing strategies will require gaining an increased knowledge of both pathogen and commensal nutrient requirements.

Members of the gut microbiota also compete with one another by producing inhibitory molecules. The bacteriocin-producing commensal Enterococcus faecalis is capable of outcompeting both endogenous enterococci species and non-bacteriocin-producing E. faecalis in mice.96 Similarly, microcin production by the probiotic E. coli strain Nissle 1917 limits colonization of pathogenic Enterobacteriaceae species, including adherent-invasive E. coli and S. enterica Typhi, in a mouse model of gut inflammation.97 Lastly, a study in mice suggests that resistance to C. difficile colonization may be mediated by the production of secondary bile acids by Clostridium scindens.91 Gut microbes, including C. scindens, convert primary bile acids made by the host to modified, secondary bile acids. Some of these microbial metabolites, including deoxycholic acid (DCA) and lithocholic acid (LCA), inhibit the growth of vegetative C. difficile cells.95 When antibiotic-treated mice were administered C. scindens alone or in combination with three additional bacteria prior to challenge with C. difficile, both groups of mice had significantly lower burden of C. difficile, less weight loss, and decreased mortality. Both the relative amount of secondary bile acids in intestinal contents as well as the abundance of the secondary bile acid biosynthesis genes correlated significantly with resistance to C. difficile, suggesting a possible


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mechanism of action. These intriguing studies demonstrate that single bacterial species capable of producing inhibitory molecules can profoundly affect pathogen colonization. Use of probiotic strains that produce such metabolites (via native or engineered pathways), or directly administering these molecules, could provide a means of preventing infection without greatly altering the resident microbiota.

Finally, members of the gut microbiota can negatively influence virulence in pathogens, both on their own and in conjunction with alterations in diet. Feeding mice a high-fiber diet was linked to significantly increased morbidity and mortality after infection with STEC. This association is thought to result from increased butyrate production by commensal microbes, which increases the expression of the shiga toxin receptor on host epithelial cells.98 In a study examining the human gut microbiota during and after Vibrio cholerae infection, the commensal Ruminococcus obeum was strongly associated with recovery.99 The ability of R. obeum to restrict pathogen colonization results from production of the small molecule autoinducer-2, which disrupts quorum sensing in V. cholerae, repressing expression of several virulence factors. Additionally, the secondary bile acid deoxycholic acid, which is produced by several species of Bifidobacterium and Clostridium, inhibits the V. cholerae type VI secretion system.100 These findings highlight the need to gain a molecular understanding of gut microbe-pathogen interactions and further suggest that gut microbiota manipulation represent a promising avenue for preventing colonization and infection by pathogens.

Outlook and Conclusion



Research on the human microbiota is undergoing an exciting transition as we move from an era of observational and taxonomical descriptions of these communities to investigations that aim to decipher the biological functions of these organisms and the mechanisms by which they contribute to health and disease. A limited but growing body of research suggests that various human-associated microbial communities can affect infection and disease progression in the context of the global health threats of tuberculosis, malaria, HIV, and enteric infections. Initial insights into the mechanistic links between the human microbiota and infectious disease have revealed new opportunities for therapeutic intervention, with a central finding being the effects of microbiota-immune system interactions on infection risk. A more thorough understanding of these complex interactions, including identifying which members of the microbiota reduce or increase inflammation and characterizing the molecular mechanisms underlying these effects, will inform future microbiota-based therapies.

Chemists and chemical biologists can contribute significantly in this area.101 It is clear that there is an enormous gap in our understanding of metabolic functions of the human microbiota both from a genetic as well as a biochemical perspective.102 Chemical knowledge can play a critical role in linking microbial activities to genes and enzymes.103 The development and use of small molecule probes may help to interrogate microbiota-immune system interactions at the signaling pathway level. The identification, isolation and structural characterization of individual microbial factors that influence pathogens or the host immune system represents another exciting objective.104


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In order to fully realize the potential of microbiota-targeted strategies for treating and preventing infectious disease, we will also need to address the major challenges of manipulating the composition and function of these complex communities.105 Chemists can help to develop these next-generation approaches for microbiota manipulation, including narrow-spectrum antibiotics and inhibitors of specific microbial activities. In addition to serving as tools, such molecules may be candidates for therapeutic development. Overall, our increasing knowledge of the mechanistic links behind microbiota-infectious disease associations, coupled with emerging strategies for manipulating microbiotas, promises to reveal and enable novel interventions to combat the major global health threat of infectious disease.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

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Table of Contents Graphic


Page 50 of 57

Page 51 of 57


Lungs 1 Tuberculosis 2 Gastrointestinal Tract 3 Tuberculosis Malaria 4 Enteric/Diarrheal Disease 5 Penis & Vagina HIV 6 7 8 9 Midgut 10 Malaria 11 Skin ACS Paragon Plus Environment 12 Malaria 13 14

A

ACS Infectious Diseases HO

OH OH

1 2 3 4 5 6B 7 8 9 10 11 12 13 C 14 15 16 17 18 19 20 21 D 22 23 24 25 26 27 28 29 30

OH O

HO

HO

OH

Growth substrate

OH

O

O HO

HO

OH OH

OH O

HO

OH

O

HO

Expansion of beneficial organisms

O

Page 52 of 57

O HO

OH

O

HO

O

O

OH

Fructooligosaccharides

OH

n = 0-6

O HO

Lactulose

OH

OH

Natural strains

Strains or consortia

Engineered probiotic anti-Plasmodium effectors e.g. lytic peptide (Shiva1)

Introduction of new organisms

Lactobacillus plantarum

Bifidobacterium infantis

Pantoea agglomerans

O O HO H N

R

OH O

O

OH

Selectively targets E. coli or P. aeruginosa

S

O

or

N O

Removal of microbes and associated functions

O

HO

Broad or narrow spectum activity

Fidaxomicin

Cl

O

Targets Clostridium difficile

‘Phagoburn’ phage cocktail

OH

OH

O

O

O OMe

OH

O

Cl O

OH

HO H N

H N

A

OH

N

A

S

S

OH OH

HN

HN

O

Non-lethal inhibitors

B

Inhibition of specific microbial functions

O

O

O

O

HO

O

ACS Paragon Plus Environment O

OH

+

CO2H

N N

SN-38G Deactivated, glucuronidated drug

OH

O N

OH OH

CO2H

HO

O N

B

O

OH O

Gut bacterial β-glucuronidases

N O O

OH

SN-38 Topoisomerase inhibitor

A Page 53 of 57 ACS Infectious Diseases Control lung

Anaerobic genera

TB-susceptible lung Anaerobic genera

1Butyrate production Butyrate production 2 INF-γ INF-γ 3 IL-17A IL-17A 4 5 6 7 8 9 Anaerobic genera 10 B Prevotella 11 Veillonella 12 Haemophilus 13 14 Carbohydrate fermentation 15 O 16 Butyrate OH 17 18 INF-γ Response 19CD8 ACS Paragon Plus IL-17A Environment to TB? 20 Pro-inflammatory CD4 21 cytokines +

+

ACS Infectious Diseases Page 54 of 57 Increased bite frequency?

1 2 3 4 1 2 3 5 6 7 4 5 6 8 Volatile attractive 9 compounds 10 Human skin 11 ACS Paragon Plus Environment 12 13 14 O

O

S

H

O

O

O

OH

O

HO

S

Page A 55 of 57

Anaerobic genera

Penile surface

1 2 3 4 5 6 7 8 9 10 C 11 12 13 14 15 16 17

Inflammation IL-8

Th17

HIV-susceptible cells

CD4+

HO

P

O

O

Inflammation CD4+

HIV-susceptible cells

Increased HIV infection risk

NH2

Gardnerella vaginalis

N

N

Tenofovir

Diversity

Not dominated by Lactobacillus

Vaginal lumen

Increased HIV infection risk

N O

ACS Infectious B Diseases

N

vitro Environment ACS ParagoninPlus

HIV preventative treatment

Unknown genes and enzymes

NH 2 N N H

N N

Adenine

+

Uncharacterized products

A

Competition for nutrients ACS Infectious Diseases OH

OH OH

O

HO

OH HO

O

HO OH

Page 56 of 57 O

OH

OH OH 1 OH OH E. coli HS 2 OH OH + OH O E. coli EDL933 O HO 3 OH HO OH HO HO (STEC) NH 4 O 5 6 Preferred carbon sources E. coli Nissle 1917 7 8B Direct inhibition 9 Ribosomally10 synthesized posttranslationally 11 modified peptides 12 (bacteriocins, Gut microbes Gut microbes microcins) 13 Pathogens (Clostridium (E. faecalis Secondary bile acids 14scindens) E. coli) (deoxycholic acid) 15 16 Altered gene expression Attenuating virulence C HO OH 17 B 18 Virulence ACS Paragon Plus Environment HO factors 19 O HO Ruminococcus obeum VIbrio cholerae 20 AI-2 21 O

OH

OH

H

H

H

HO

H

H

GLOBAL HEALTH

Page 57 of 57 ACS Infectious Diseases TB

1 2 3 4 ENTERIC 5 DISEASE

MALARIA

ACS Paragon Plus Environment HIV

The Human Microbiota, Infectious Disease, and Global Health

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