Control of lysogeny and antiphage defense by a prophage-encoded kinase-phosphatase module.

The filamentous 'Pf' bacteriophages of Pseudomonas aeruginosa play roles in biofilm formation and virulence, but mechanisms governing Pf prophage activation in biofilms are unclear. Here, we identify a prophage regulatory module, KKP (kinase-kinase-phosphatase), that controls virion production of co-resident Pf prophages and mediates host defense against diverse lytic phages. KKP consists of Ser/Thr kinases PfkA and PfkB, and phosphatase PfpC. The kinases have multiple host targets, one of which is MvaU, a host nucleoid-binding protein and known prophage-silencing factor. Characterization of KKP deletion and overexpression strains with transcriptional, protein-level and prophage-based approaches indicates that shifts in the balance between kinase and phosphatase activities regulate phage production by controlling MvaU phosphorylation. In addition, KKP acts as a tripartite toxin-antitoxin system that provides defense against some lytic phages. A conserved lytic phage replication protein inhibits the KKP phosphatase PfpC, stimulating toxic kinase activity and blocking lytic phage production. Thus, KKP represents a phosphorylation-based mechanism for prophage regulation and antiphage defense. The conservation of KKP gene clusters in >1000 diverse temperate prophages suggests that integrated control of temperate and lytic phage infection by KKP-like regulatory modules may play a widespread role in shaping host cell physiology.

A conserved interaction between the effector Sca4 and host endocytic machinery suggests additional roles for Sca4 during rickettsial infection.

Intracellular bacterial pathogens deploy secreted effector proteins that manipulate diverse host machinery and pathways to promote infection. Although many effectors carry out a single specific function or interaction, there are a growing number of secreted pathogen effectors capable of interacting with multiple host factors. However, few effectors secreted by obligate intracellular Rickettsia species have been linked to multiple host targets. Here, we investigated the conserved rickettsial secreted effector Sca4, which was previously shown to interact with host vinculin to promote cell-to-cell spread in the model Rickettsia species R. parkeri . We discovered that Sca4 also binds the host cell endocytic factor clathrin heavy chain (CHC, CLTC ) via a conserved segment in the Sca4 N-terminus. Ablation of CLTC expression or chemical inhibition of endocytosis reduced R. parkeri cell-to-cell spread, indicating that clathrin promotes efficient spread between mammalian cells. This activity was independent of Sca4 and appeared restricted to the recipient host cell, suggesting that the Sca4-clathrin interaction also regulates another aspect of the infectious lifecycle. Indeed, R. parkeri lacking Sca4 or expressing a Sca4 truncation unable to bind clathrin had markedly reduced burdens in tick cells, hinting at a cell-type specific function for the Sca4-clathrin interaction. Sca4 homologs from diverse Rickettsia species also bound clathrin, suggesting that the function of this novel effector-host interaction may be broadly important for rickettsial infection. We conclude that Sca4 has multiple targets during infection and that rickettsiae may manipulate host endocytic machinery to facilitate several stages of their life cycles.

Erratum: A Bacterial Pathogen Senses Host Mannose to Coordinate Virulence.

[This corrects the article DOI: 10.1016/j.isci.2019.09.028.].

Pathogenic Rickettsia spp. as emerging models for bacterial biology.

Our understanding of free-living bacterial models like Escherichia coli far outpaces that of obligate intracellular bacteria, which cannot be cultured axenically. All obligate intracellular bacteria are host-associated, and many cause serious human diseases. Their constant exposure to the distinct biochemical niche of the host has driven the evolution of numerous specialized bacteriological and genetic adaptations, as well as innovative molecular mechanisms of infection. Here, we review the history and use of pathogenic Rickettsia species, which cause an array of vector-borne vascular illnesses, as model systems to probe microbial biology. Although many challenges remain in our studies of these organisms, the rich pathogenic and biological diversity of Rickettsia spp. constitutes a unique backdrop to investigate how microbes survive and thrive in host and vector cells. We take a bacterial-focused perspective and highlight emerging insights that relate to new host-pathogen interactions, bacterial physiology, and evolution. The transformation of Rickettsia spp. from pathogens to models demonstrates how recalcitrant microbes may be leveraged in the lab to tap unmined bacterial diversity for new discoveries. Rickettsia spp. hold great promise as model systems not only to understand other obligate intracellular pathogens but also to discover new biology across and beyond bacteria.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Intelectin-1 binds and alters the localization of the mucus barrier-modifying bacterium Akkermansia muciniphila.

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host-microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell-mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.

Undecaprenyl phosphate translocases confer conditional microbial fitness.

The microbial cell wall is essential for maintenance of cell shape and resistance to external stressors1. The primary structural component of the cell wall is peptidoglycan, a glycopolymer with peptide crosslinks located outside of the cell membrane1. Peptidoglycan biosynthesis and structure are responsive to shifting environmental conditions such as pH and salinity2-6, but the mechanisms underlying such adaptations are incompletely understood. Precursors of peptidoglycan and other cell surface glycopolymers are synthesized in the cytoplasm and then delivered across the cell membrane bound to the recyclable lipid carrier undecaprenyl phosphate7 (C55-P, also known as UndP). Here we identify the DUF368-containing and DedA transmembrane protein families as candidate C55-P translocases, filling a critical gap in knowledge of the proteins required for the biogenesis of microbial cell surface polymers. Gram-negative and Gram-positive bacteria lacking their cognate DUF368-containing protein exhibited alkaline-dependent cell wall and viability defects, along with increased cell surface C55-P levels. pH-dependent synthetic genetic interactions between DUF368-containing proteins and DedA family members suggest that C55-P transporter usage is dynamic and modulated by environmental inputs. C55-P transporter activity was required by the cholera pathogen for growth and cell shape maintenance in the intestine. We propose that conditional transporter reliance provides resilience in lipid carrier recycling, bolstering microbial fitness both inside and outside the host.

Emerging Concepts in Cholera Vaccine Design.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae and constitutes a significant public health threat in many areas of the world. V. cholerae infection elicits potent and long-lasting immunity, and efforts to develop cholera vaccines have been ongoing for more than a century. Currently available inactivated two-dose oral cholera vaccines are increasingly deployed to both prevent and actively curb cholera outbreaks, and they are key components of the global effort to eradicate cholera. However, these killed whole-cell vaccines have several limitations, and a variety of new oral and nonoral cholera vaccine platforms have recently been developed. Here, we review emerging concepts in cholera vaccine design and implementation that have been driven by insights from human and animal studies. As a prototypical vaccine-preventable disease, cholera continues to be an excellent target for the development and application of cutting-edge technologies and platforms that may transform vaccinology.

Genomic and Phenotypic Insights for Toxigenic Clinical Vibrio cholerae O141.

Vibrio cholerae remains a major public health threat worldwide, causing millions of cholera cases each year. Although much is known about the evolution and pathogenicity of the O1/O139 serogroups of V. cholerae, information is lacking on the molecular epidemiology of non‒O1/O139 strains isolated from patients who have diarrheal illnesses. We performed whole-genome sequence analysis and in vivo infections to investigate characteristics of V. cholerae O141 isolated from sporadic diarrheal cases in 4 countries. The strains formed a distinct phylogenetic clade distinguishable from other serogroups and a unique multilocus sequence type 42, but interstrain variation suggests that O141 isolates are not clonal. These isolates encode virulence factors including cholera toxin and the toxin-coregulated pilus, as well as a type 3 secretion system. They had widely variable capacities for intestinal colonization in the infant mouse model. We propose that O141 isolates comprise a distinct clade of V. cholerae non‒O1/O139, and their continued surveillance is warranted.

Animal models for dissecting Vibrio cholerae intestinal pathogenesis and immunity.

The human diarrheal disease cholera is caused by the bacterium Vibrio cholerae. Efforts to develop animal models that closely mimic cholera to study the pathogenesis of this disease began >125 years ago. Here, we review currently used non-surgical, oral inoculation-based animal models for investigation of V. cholerae intestinal colonization and disease and highlight recent discoveries that have illuminated mechanisms of cholera pathogenesis and immunity, particularly in the area of how V. cholerae interacts with the gut microbiome to influence infection. The emergence of high-throughput tools for studies of pathogen-host interactions, along with continued advances in host genetic engineering and manipulation in animal models of V. cholerae will deepen understanding of cholera pathogenesis, uncovering knowledge important for control of this globally important bacterial pathogen.

Proteomic analysis of the host-pathogen interface in experimental cholera.

The microbial cell surface is a site of critical microbe-host interactions that often control infection outcomes. Defining the set of host proteins present at this interface has been challenging. Here we used a surface-biotinylation approach coupled to quantitative mass spectrometry to identify and quantify both bacterial and host proteins present on the surface of diarrheal fluid-derived Vibrio cholerae in an infant rabbit model of cholera. The V. cholerae surface was coated with numerous host proteins, whose abundance were driven by the presence of cholera toxin, including the C-type lectin SP-D. Mice lacking SP-D had enhanced V. cholerae intestinal colonization, and SP-D production shaped both host and pathogen transcriptomes. Additional host proteins (AnxA1, LPO and ZAG) that bound V. cholerae were also found to recognize distinct taxa of the murine intestinal microbiota, suggesting that these host factors may play roles in intestinal homeostasis in addition to host defense.

Actinobacillus utilizes a binding protein-dependent ABC transporter to acquire the active form of vitamin B6.

Bacteria require high-efficiency uptake systems to survive and proliferate in nutrient-limiting environments, such as those found in host organisms. ABC transporters in the bacterial plasma membrane provide a mechanism for transport of many substrates. In this study, we examine an operon containing a periplasmic binding protein in Actinobacillus for its potential role in nutrient acquisition. The electron density map of 1.76 Å resolution obtained from the crystal structure of the periplasmic binding protein was best fit with a molecular model containing a pyridoxal-5'-phosphate (P5P/pyridoxal phosphate/the active form of vitamin B6) ligand within the protein's binding site. The identity of the P5P bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from Actinobacillus pleuropneumoniae into an Escherichia coli K-12 strain that was devoid of a key enzyme required for P5P synthesis. The growth of this strain at low levels of P5P supports the functional role of this operon in P5P uptake. This is the first report of a dedicated P5P bacterial uptake system, but through bioinformatics, we discovered homologs mainly within pathogenic representatives of the Pasteurellaceae family, suggesting that this operon exists more widely outside the Actinobacillus genus.

BipA exerts temperature-dependent translational control of biofilm-associated colony morphology in Vibrio cholerae.

Adaptation to shifting temperatures is crucial for the survival of the bacterial pathogen Vibrio cholerae. Here, we show that colony rugosity, a biofilm-associated phenotype, is regulated by temperature in V. cholerae strains that naturally lack the master biofilm transcriptional regulator HapR. Using transposon-insertion mutagenesis, we found the V. cholerae ortholog of BipA, a conserved ribosome-associated GTPase, is critical for this temperature-dependent phenomenon. Proteomic analyses revealed that loss of BipA alters the synthesis of >300 proteins in V. cholerae at 22°C, increasing the production of biofilm-related proteins including the key transcriptional activators VpsR and VpsT, as well as proteins important for diverse cellular processes. At low temperatures, BipA protein levels increase and are required for optimal ribosome assembly in V. cholerae, suggesting that control of BipA abundance is a mechanism by which bacteria can remodel their proteomes. Our study reveals a remarkable new facet of V. cholerae's complex biofilm regulatory network.

Dissecting serotype-specific contributions to live oral cholera vaccine efficacy.

The O1 serogroup of Vibrio cholerae causes pandemic cholera and is divided into the Ogawa and Inaba serotypes. The O-antigen is V. cholerae's immunodominant antigen, and the two serotypes, which differ by the presence or absence of a terminally methylated O-antigen, likely influence development of immunity to cholera and oral cholera vaccines (OCVs). However, there is no consensus regarding the relative immunological potency of each serotype, in part because previous studies relied on genetically heterogeneous strains. Here, we engineered matched serotype variants of a live OCV candidate, HaitiV, and used a germfree mouse model to evaluate the immunogenicity and protective efficacy of each vaccine serotype. By combining vibriocidal antibody quantification with single- and mixed-strain infection assays, we found that all three HaitiV variants-InabaV, OgawaV, and HikoV (bivalent Inaba/Ogawa)-were immunogenic and protective. None of the vaccine serotypes were superior across both of these vaccine metrics, suggesting that the impact of O1-serotype variation in OCV design, although detectable, is subtle. However, all three live vaccines significantly outperformed formalin-killed HikoV, supporting the idea that live OCV usage will bolster current cholera control practices. The potency of OCVs was found to be challenge strain-dependent, emphasizing the importance of appropriate strain selection for cholera challenge studies. Our findings and experimental approaches will be valuable for guiding the development of live OCVs and oral vaccines for additional pathogens.

Increased Listeria monocytogenes Dissemination and Altered Population Dynamics in Muc2-Deficient Mice.

The mucin Muc2 is a major constituent of the mucus layer that covers the intestinal epithelium and creates a barrier between epithelial cells and luminal commensal or pathogenic microorganisms. The Gram-positive foodborne pathogen Listeria monocytogenes can cause enteritis and also disseminate from the intestine to give rise to systemic disease. L. monocytogenes can bind to intestinal Muc2, but the influence of the Muc2 mucin barrier on L. monocytogenes intestinal colonization and systemic dissemination has not been explored. Here, we used an orogastric L. monocytogenes infection model to investigate the role of Muc2 in host defense against L. monocytogenes Compared to wild-type mice, we found that Muc2-/- mice exhibited heightened susceptibility to orogastric challenge with L. monocytogenes, with higher mortality, elevated colonic pathology, and increased pathogen burdens in both the intestinal tract and distal organs. In contrast, L. monocytogenes burdens were equivalent in wild-type and Muc2-/- animals when the pathogen was administered intraperitoneally, suggesting that systemic immune defects related to Muc2 deficiency do not explain the heightened pathogen dissemination observed in oral infections. Using a barcoded L. monocytogenes library to measure intrahost pathogen population dynamics, we found that Muc2-/- animals had larger pathogen founding population sizes in the intestine and distal sites than observed in wild-type animals. Comparisons of barcode frequencies suggested that the colon becomes the major source for seeding the internal organs in Muc2-/- animals. Together, our findings reveal that Muc2 mucin plays a key role in controlling L. monocytogenes colonization, dissemination, and population dynamics.

Type I interferon remodels lysosome function and modifies intestinal epithelial defense.

Organelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I IFN (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity, and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including IFITM3, SLC15A3, and CNP contribute to lysosome acidification. IFN-I-dependent lysosome acidification was associated with elevated intracellular Salmonella virulence gene expression, rupture of the Salmonella-containing vacuole, and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium where Salmonella initiates infection, indicating that IFN-I signaling can modify innate defense in the epithelial compartment. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.

Genetic Dissection of the Fermentative and Respiratory Contributions Supporting Vibrio cholerae Hypoxic Growth.

Both fermentative and respiratory processes contribute to bacterial metabolic adaptations to low oxygen tension (hypoxia). In the absence of O2 as a respiratory electron sink, many bacteria utilize alternative electron acceptors, such as nitrate (NO3-). During canonical NO3- respiration, NO3- is reduced in a stepwise manner to N2 by a dedicated set of reductases. Vibrio cholerae, the etiological agent of cholera, requires only a single periplasmic NO3- reductase (NapA) to undergo NO3- respiration, suggesting that the pathogen possesses a noncanonical NO3- respiratory chain. In this study, we used complementary transposon-based screens to identify genetic determinants of general hypoxic growth and NO3- respiration in V. cholerae We found that while the V. cholerae NO3- respiratory chain is primarily composed of homologues of established NO3- respiratory genes, it also includes components previously unlinked to this process, such as the Na+-NADH dehydrogenase Nqr. The ethanol-generating enzyme AdhE was shown to be the principal fermentative branch required during hypoxic growth in V. cholerae Relative to single adhE or napA mutant strains, a V. cholerae strain lacking both genes exhibited severely impaired hypoxic growth in vitro and in vivo Our findings reveal the genetic basis of a specific interaction between disparate energy production pathways that supports pathogen fitness under shifting conditions. Such metabolic specializations in V. cholerae and other pathogens are potential targets for antimicrobial interventions.IMPORTANCE Bacteria reprogram their metabolism in environments with low oxygen levels (hypoxia). Typically, this occurs via regulation of two major, but largely independent, metabolic pathways: fermentation and respiration. In this study, we found that the diarrheal pathogen Vibrio cholerae has a respiratory chain for NO3- that consists largely of components found in other NO3- respiratory systems but also contains several proteins not previously linked to this process. Both AdhE-dependent fermentation and NO3- respiration were required for efficient pathogen growth under both laboratory conditions and in an animal infection model. These observations provide a specific example of fermentative respiratory interactions and identify metabolic vulnerabilities that may be targetable for new antimicrobial agents in V. cholerae and related pathogens.

Transient Intestinal Colonization by a Live-Attenuated Oral Cholera Vaccine Induces Protective Immune Responses in Streptomycin-Treated Mice.

Current mouse models for evaluating the efficacy of live oral cholera vaccines (OCVs) have important limitations. Conventionally raised adult mice are resistant to intestinal colonization by Vibrio cholerae, but germfree mice can be colonized and have been used to study OCV immunogenicity. However, germfree animals have impaired immune systems and intestinal physiology; also, live OCVs colonize germfree mice for many months, which does not mimic the clearance kinetics of live OCVs in humans. In this study, we leveraged antibiotic-treated, conventionally raised adult mice to study the effects of transient intestinal colonization by a live OCV V. cholerae strain. In a single-dose vaccination regimen, we found that HaitiV, a live-attenuated OCV candidate, was cleared by streptomycin-treated adult mice within 2 weeks after oral inoculation. This transient colonization elicited far stronger adaptive immune correlates of protection against cholera than did inactivated whole-cell HaitiV. Infant mice from HaitiV-vaccinated dams were also significantly more protected from choleric disease than pups from inactivated-HaitiV-vaccinated dams. Our findings establish the benefits of antibiotic-treated mice for live-OCV studies as well as their limitations and underscore the immunogenicity of HaitiV.IMPORTANCE Oral cholera vaccines (OCVs) are being deployed to combat cholera, but current killed OCVs require multiple doses and show little efficacy in young children. Live OCVs have the potential to overcome these limitations, but small-animal models for testing OCVs have shortcomings. We used an antibiotic treatment protocol for conventional adult mice to study the effects of short-term colonization by a single dose of HaitiV, a live-OCV candidate. Vaccinated mice developed vibriocidal antibodies against V. cholerae and delivered pups that were resistant to cholera, whereas mice vaccinated with inactivated HaitiV did not. These findings demonstrate HaitiV's immunogenicity and suggest that this antibiotic treatment protocol will be useful for evaluating the efficacy of live OCVs.

A Bacterial Pathogen Senses Host Mannose to Coordinate Virulence.

Bacterial pathogens are thought to activate expression of virulence genes upon detection of host-associated cues, but identification of the nature of such signals has proved difficult. We generated a genome-scale defined transposon mutant library in Edwardsiella piscicida, an important fish pathogen, to quantify the fitness of insertion mutants for intracellular growth in macrophages and in turbot (Scophthalmus maximus). These screens identified EvrA, a transcription activator that induces expression of esrB, a key virulence regulator. EvrA is directly bound and activated by mannose-6-phosphate (man-6P) derived from actively imported mannose. Mutants lacking EvrA or expressing an EvrA unable to bind man-6P were similarly attenuated in turbot. Exogenously added mannose promoted E. piscicida virulence, and high levels of mannose were detected in fish tissue. Together, these observations reveal that binding of a host-derived sugar to a transcription factor can facilitate pathogen sensing of the host environment and trigger virulence programs.

Oral immunization with a probiotic cholera vaccine induces broad protective immunity against Vibrio cholerae colonization and disease in mice.

Oral cholera vaccines (OCVs) are being increasingly employed, but current killed formulations generally require multiple doses and lack efficacy in young children. We recently developed a new live-attenuated OCV candidate (HaitiV) derived from a Vibrio cholerae strain isolated during the 2010 Haiti cholera epidemic. HaitiV exhibited an unexpected probiotic-like activity in infant rabbits, preventing intestinal colonization and disease by wild-type V. cholerae before the onset of adaptive immunity. However, it remained unknown whether HaitiV would behave similarly to other OCVs to stimulate adaptive immunity against V. cholerae. Here, we orally immunized adult germ-free female mice to test HaitiV's immunogenicity. HaitiV safely and stably colonized vaccinated mice and induced known adaptive immune correlates of cholera protection within 14 days of administration. Pups born to immunized mice were protected against lethal challenges of both homologous and heterologous V. cholerae strains. Cross-fostering experiments revealed that protection was not dependent on vaccine colonization in or transmission to the pups. These findings demonstrate the protective immunogenicity of HaitiV and support its development as a new tool for limiting cholera.