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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Mammalian, non-mammalian, and invertebrate species are currently used to model Vibrio cholerae pathogenicity. • Models vary considerably in their recapitulation of V. cholerae- host interactions in the human intestine. • Recent work in animals has revealed molecular details of the host- V. cholerae -microbiome axis. • Open areas for investigation include intestinal innate defense against V. cholerae and determinants of cholera transmission. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 AdhEdependent 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Type I interferons (IFNs) play a critical role in antiviral immune responses, but can be deleterious to the host during some bacterial infections. Listeria monocytogenes ( Lm) induces a type I IFN response by activating cytosolic antiviral surveillance pathways. This is beneficial to the bacteria as mice lacking the type I IFN receptor (IFNAR1−/−) are resistant to systemic infection by Lm. The mechanisms by which type I IFNs promote Lm infection are unclear. Here, we show that IFNAR1 is required for dissemination of Lm within infection foci in livers of infected mice and for efficient cell-to-cell spread in vitro in macrophages. IFNAR1 promotes ActA polarization and actin-based motility in the cytosol of host cells. Our studies suggest type I IFNs directly impact the intracellular life cycle of Lm and provide new insight into the mechanisms used by bacterial pathogens to exploit the type I IFN response. [ABSTRACT FROM AUTHOR]

Efficient acquisition of extracellular nutrients is essential for bacterial pathogenesis, however the identities and mechanisms for transport of many of these substrates remain unclear. Here, we investigate the predicted iron-binding transporter AfuABC and its role in bacterial pathogenesis in vivo. By crystallographic, biophysical and in vivo approaches, we show that AfuABC is in fact a cyclic hexose/heptose-phosphate transporter with high selectivity and specificity for a set of ubiquitous metabolites (glucose-6-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate). AfuABC is conserved across a wide range of bacterial genera, including the enteric pathogens EHEC O157:H7 and its murine-specific relative Citrobacter rodentium, where it lies adjacent to genes implicated in sugar sensing and acquisition. C. rodentium ΔafuA was significantly impaired in an in vivo murine competitive assay as well as its ability to transmit infection from an afflicted to a naïve murine host. Sugar-phosphates were present in normal and infected intestinal mucus and stool samples, indicating that these metabolites are available within the intestinal lumen for enteric bacteria to import during infection. Our study shows that AfuABC-dependent uptake of sugar-phosphates plays a critical role during enteric bacterial infection and uncovers previously unrecognized roles for these metabolites as important contributors to successful pathogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Background and purpose: The unfolded protein response (UPR) is activated in response to hypoxia-induced stress in the endoplasmic reticulum (ER) and consists of three distinct signaling arms. Here we explore the potential of targeting two of these arms with new potent small-molecule inhibitors designed against IRE1α and PERK. Methods: We utilized shRNAs and small-molecule inhibitors of IRE1α (4μ8c) and PERK (GSK-compound 39). XBP1 splicing and DNAJB9 mRNA was measured by qPCR and was used to monitor IRE1α activity. PERK activity was monitored by immunoblotting eIF2α phosphorylation and qPCR of DDIT3 mRNA. Hypoxia tolerance was measured using proliferation and clonogenic cell survival assays of cells exposed to mild or severe hypoxia in the presence of the inhibitors. Results: Using knockdown experiments we show that PERK is essential for survival of KP4 cells while knockdown of IRE1α dramatically decreases the proliferation and survival of HCT116 during hypoxia. Further, we show that in response to both hypoxia and other ER stress-inducing agents both 4μ8c and the PERK inhibitor are selective and potent inhibitors of IRE1α and PERK activation, respectively. However, despite potent inhibition of IRE1α activation, 4μ8c had no effect on cell proliferation or clonogenic survival of cells exposed to hypoxia. This was in contrast to the inactivation of PERK signaling with the PERK inhibitor, which reduced tolerance to hypoxia and other ER stress inducing agents. Conclusions: Our results demonstrate that IRE1α but not its splicing activity is important for hypoxic cell survival. The PERK signaling arm is uniquely important for promoting adaptation and survival during hypoxia-induced ER stress and should be the focus of future therapeutic efforts. [Copyright &y& Elsevier]

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-50-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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • ER stress in intestinal epithelial cells (IEC) drives gut Th17 differentiation • Th17 cells induced by IEC-ER stress require H 2 O 2 generated by DUOX2/DUOXA2 • IEC-ROS induce Th17 differentiation through purine metabolites, including xanthine • IEC-ER stress drives microbial Th17 induction even under germ-free conditions Intestinal epithelial cell (IEC)-adherent microbes are important for generation of intestinal Th17 cells. Duan et al. report that microbial adhesion drives IEC-ER stress and unfolded protein response (UPR). The UPR enhances IEC production of reactive oxygen species and purine metabolites that subsequently promote Th17 differentiation in the gut. [ABSTRACT FROM AUTHOR]

Random spanning trees of a graph G are governed by a corresponding probability mass distribution (or "law"), μ , defined on the set of all spanning trees of G. This paper addresses the problem of choosing μ in order to utilize the edges as "fairly" as possible. This turns out to be equivalent to minimizing, with respect to μ , the expected overlap of two independent random spanning trees sampled with law μ. In the process, we introduce the notion of homogeneous graphs. These are graphs for which it is possible to choose a random spanning tree so that all edges have equal usage probability. The main result is a deflation process that identifies a hierarchical structure of arbitrary graphs in terms of homogeneous subgraphs, which we call homogeneous cores. A key tool in the analysis is the spanning tree modulus, for which there exists an algorithm based on minimum spanning tree algorithms, such as Kruskal's or Prim's. [ABSTRACT FROM AUTHOR]