Enterohemorrhagic Escherichia coli responds to gut microbiota metabolites by altering metabolism and activating stress responses.

Enterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC's ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC's response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes.

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1-D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2 µg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.

Potent transmission-blocking monoclonal antibodies from naturally exposed individuals target a conserved epitope on Plasmodium falciparum Pfs230.

Pfs230 is essential for Plasmodium falciparum transmission to mosquitoes and is the protein targeted by the most advanced malaria-transmission-blocking vaccine candidate. Prior understanding of functional epitopes on Pfs230 is based on two monoclonal antibodies (mAbs) with moderate transmission-reducing activity (TRA), elicited from subunit immunization. Here, we screened the B cell repertoire of two naturally exposed individuals possessing serum TRA and identified five potent mAbs from sixteen Pfs230 domain-1-specific mAbs. Structures of three potent and three low-activity antibodies bound to Pfs230 domain 1 revealed four distinct epitopes. Highly potent mAbs from natural infection recognized a common conformational epitope that is highly conserved across P. falciparum field isolates, while antibodies with negligible TRA derived from natural infection or immunization recognized three distinct sites. Our study provides molecular blueprints describing P. falciparum TRA, informed by contrasting potent and non-functional epitopes elicited by natural exposure and vaccination.

Gastric acid and escape to systemic circulation represent major bottlenecks to host infection by Citrobacter rodentium.

The gastrointestinal (GI) environment plays a critical role in shaping enteric infections. Host environmental factors create bottlenecks, restrictive events that reduce the genetic diversity of invading bacterial populations. However, the identity and impact of bottleneck events on bacterial infection are largely unknown. We used Citrobacter rodentium infection of mice, a model of human pathogenic Escherichia coli infections, to examine bacterial population dynamics and quantify bottlenecks to host colonization. Using Sequence Tag-based Analysis of Microbial Populations (STAMP) we characterized the founding population size (Nb') and relatedness of C. rodentium populations at relevant tissue sites during early- and peak-infection. We demonstrate that the GI environment severely restricts the colonizing population, with an average Nb' of only 12-43 lineages (of 2,000+ inoculated) identified regardless of time or biogeographic location. Passage through gastric acid and escape to the systemic circulation were identified as major bottlenecks during C. rodentium colonization. Manipulating such events by increasing gastric pH dramatically increased intestinal Nb'. Importantly, removal of the stomach acid barrier had downstream consequences on host systemic colonization, morbidity, and mortality. These findings highlight the capability of the host GI environment to limit early pathogen colonization, controlling the population of initial founders with consequences for downstream infection outcomes.

Type VI secretion systems of pathogenic and commensal bacteria mediate niche occupancy in the gut.

The type VI secretion system (T6SS) is a contractile nanomachine widely distributed among pathogenic and commensal Gram-negative bacteria. The T6SS is used for inter-bacterial competition to directly kill competing species; however, its importance during bacterial infection in vivo remains poorly understood. We report that the murine pathogen Citrobacter rodentium, used as a model for human pathogenic Escherichia coli, harbors two functional T6SSs. C. rodentium employs its T6SS-1 to colonize the murine gastrointestinal tract by targeting commensal Enterobacteriaceae. We identify VgrG1 as a C. rodentium T6SS antibacterial effector, which exhibits toxicity in E. coli. Conversely, commensal prey species E. coli Mt1B1 employs two T6SSs of its own to counter C. rodentium colonization. Collectively, these data demonstrate that the T6SS is a potent weapon during bacterial competition and is used by both invading pathogens and resident microbiota to fight for a niche in the hostile gut environment.

Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection.

Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens.

Gut microbiota-mediated protection against diarrheal infections.

BACKGROUND: The mammalian gut microbiota is a highly abundant and diverse microbial community that resides in the gastrointestinal tract. One major benefit that the gut microbiota provides to its host is colonization resistance-the ability to prevent colonization by foreign microbes, including diarrheal pathogens such as Clostridium difficile , Salmonella enterica serovar Typhimurium and diarrheagenic Escherichia coli . METHODS: We conducted a literature review of the effects of the gut microbiota on infection by diarrheal pathogens. We used PubMed to search for relevant articles published before July 2016, as well as incorporated data from our laboratory. RESULTS: The gut microbiota provides protection from diarrheal infections both by direct inhibition of pathogens and by indirect effects on host functions. Direct effects of the microbiota on diarrheal pathogens include competing for nutrients and producing metabolites that inhibit pathogen growth or virulence. Indirect effects of the gut microbiota include promoting maintenance of the gut mucosal barrier and stimulating innate and adaptive immunity. CONCLUSIONS: Human epidemiological studies and experimental infections of laboratory animals both demonstrate that antibiotic treatment can alter the gut microbial community and thereby reduce colonization resistance against diarrheal pathogens. Further research might lead to the development of next-generation probiotics that could be used to bolster colonization resistance and thus prevent travellers' diarrheal.

Analysis of bacterial survival after exposure to reactive oxygen species or antibiotics.

The redox balance in a variety of Gram-negative bacteria was explored using redox sensitive GFP (roGFP2), J. van der Heijden et al. doi:10.1016/j.freeradbiomed.2015.11.029[1]. This data article provides Supporting material to further investigate the relationship between Salmonella typhimurium survival and oxidative stress. The first set of data presented in this article, shows the percentage of surviving bacteria after exposure to hydrogen peroxide. The second set of data shows the concentration of hydrogen peroxide that was produced by S. Typhimurium in different growth phases. The last set of data shows the percentage of surviving S. Typhimurium bacteria after exposure to different antibiotics.

Mutations in Subunits of the Activating Signal Cointegrator 1 Complex Are Associated with Prenatal Spinal Muscular Atrophy and Congenital Bone Fractures.

Transcriptional signal cointegrators associate with transcription factors or nuclear receptors and coregulate tissue-specific gene transcription. We report on recessive loss-of-function mutations in two genes (TRIP4 and ASCC1) that encode subunits of the nuclear activating signal cointegrator 1 (ASC-1) complex. We used autozygosity mapping and whole-exome sequencing to search for pathogenic mutations in four families. Affected individuals presented with prenatal-onset spinal muscular atrophy (SMA), multiple congenital contractures (arthrogryposis multiplex congenita), respiratory distress, and congenital bone fractures. We identified homozygous and compound-heterozygous nonsense and frameshift TRIP4 and ASCC1 mutations that led to a truncation or the entire absence of the respective proteins and cosegregated with the disease phenotype. Trip4 and Ascc1 have identical expression patterns in 17.5-day-old mouse embryos with high expression levels in the spinal cord, brain, paraspinal ganglia, thyroid, and submandibular glands. Antisense morpholino-mediated knockdown of either trip4 or ascc1 in zebrafish disrupted the highly patterned and coordinated process of α-motoneuron outgrowth and formation of myotomes and neuromuscular junctions and led to a swimming defect in the larvae. Immunoprecipitation of the ASC-1 complex consistently copurified cysteine and glycine rich protein 1 (CSRP1), a transcriptional cofactor, which is known to be involved in spinal cord regeneration upon injury in adult zebrafish. ASCC1 mutant fibroblasts downregulated genes associated with neurogenesis, neuronal migration, and pathfinding (SERPINF1, DAB1, SEMA3D, SEMA3A), as well as with bone development (TNFRSF11B, RASSF2, STC1). Our findings indicate that the dysfunction of a transcriptional coactivator complex can result in a clinical syndrome affecting the neuromuscular system.

Exploring the redox balance inside gram-negative bacteria with redox-sensitive GFP.

Aerobic bacteria are continuously fighting potential oxidative stress due to endogenous and exogenous reactive oxygen species (ROS). To achieve this goal, bacteria possess a wide array of defenses and stress responses including detoxifying enzymes like catalases and peroxidases; however until now, the dynamics of the intra-bacterial redox balance remained poorly understood. Herein, we used redox-sensitive GFP (roGFP2) inside a variety of gram-negative bacteria to study real-time redox dynamics immediately after a challenge with hydrogen peroxide. Using this biosensor, we determined the individual contributions of catalases and peroxidases and found that each enzyme contributes more to rapid detoxification or to prolonged catalytic activity. We also found that the total catalytic power is affected by environmental conditions. Additionally, using a Salmonella strain that is devoid of detoxifying enzymes, we examined endogenous ROS production. By measuring endogenous ROS production, we assessed the role of oxidative stress in toxicity of heavy metals and antibiotics. We found that exposure to nickel induced significant oxidative stress whereas cobalt (which was previously implicated to induce oxidative stress) did not induce ROS formation. Since a turbulent debate evolves around oxidative stress as a general killing mechanism by antibiotics (aminoglycosides, fluoroquinolones and ß-lactams), we measured oxidative stress in bacteria that were challenged with these antibiotics. Our results revealed that antibiotics do not induce ROS formation in bacteria thereby disputing a role for oxidative stress as a general killing mechanism. Together, our results expose how the intra-bacterial redox balance in individual microorganisms is affected by environmental conditions and encounters with stress-inducing compounds. These findings demonstrate the significant potential of roGFP2 as a redox biosensor in gram-negative bacteria to investigate redox dynamics under a variety of circumstances.

Chemical communication in the gut: Effects of microbiota-generated metabolites on gastrointestinal bacterial pathogens.

Gastrointestinal pathogens must overcome many obstacles in order to successfully colonize a host, not the least of which is the presence of the gut microbiota, the trillions of commensal microorganisms inhabiting mammals' digestive tracts, and their products. It is well established that a healthy gut microbiota provides its host with protection from numerous pathogens, including Salmonella species, Clostridium difficile, diarrheagenic Escherichia coli, and Vibrio cholerae. Conversely, pathogenic bacteria have evolved mechanisms to establish an infection and thrive in the face of fierce competition from the microbiota for space and nutrients. Here, we review the evidence that gut microbiota-generated metabolites play a key role in determining the outcome of infection by bacterial pathogens. By consuming and transforming dietary and host-produced metabolites, as well as secreting primary and secondary metabolites of their own, the microbiota define the chemical environment of the gut and often determine specific host responses. Although most gut microbiota-produced metabolites are currently uncharacterized, several well-studied molecules made or modified by the microbiota are known to affect the growth and virulence of pathogens, including short-chain fatty acids, succinate, mucin O-glycans, molecular hydrogen, secondary bile acids, and the AI-2 quorum sensing autoinducer. We also discuss challenges and possible approaches to further study of the chemical interplay between microbiota and gastrointestinal pathogens.

Frequency and phenotypic spectrum of germline mutations in POLE and seven other polymerase genes in 266 patients with colorectal adenomas and carcinomas.

In a number of families with colorectal adenomatous polyposis or suspected Lynch syndrome/HNPCC, no germline alteration in the APC, MUTYH, or mismatch repair (MMR) genes are found. Missense mutations in the polymerase genes POLE and POLD1 have recently been identified as rare cause of multiple colorectal adenomas and carcinomas, a condition termed polymerase proofreading-associated polyposis (PPAP). The aim of the present study was to evaluate the clinical relevance and phenotypic spectrum of polymerase germline mutations. Therefore, targeted sequencing of the polymerase genes POLD1, POLD2, POLD3, POLD4, POLE, POLE2, POLE3 and POLE4 was performed in 266 unrelated patients with polyposis or fulfilled Amsterdam criteria. The POLE mutation c.1270C>G;p.Leu424Val was detected in four unrelated patients. The mutation was present in 1.5% (4/266) of all patients, 4% (3/77) of all familial cases and 7% (2/30) of familial polyposis cases. The colorectal phenotype in 14 affected individuals ranged from typical adenomatous polyposis to a HNPCC phenotype, with high intrafamilial variability. Multiple colorectal carcinomas and duodenal adenomas were common, and one case of duodenal carcinoma was reported. Additionally, various extraintestinal lesions were evident. Nine further putative pathogenic variants were identified. The most promising was c.1306C>T;p.Pro436Ser in POLE. In conclusion, a PPAP was identified in a substantial number of polyposis and familial colorectal cancer patients. Screening for polymerase proofreading mutations should therefore be considered, particularly in unexplained familial cases. The present study broadens the phenotypic spectrum of PPAP to duodenal adenomas and carcinomas, and identified novel, potentially pathogenic variants in four polymerase genes.

Genome-wide CNV analysis in 221 unrelated patients and targeted high-throughput sequencing reveal novel causative candidate genes for colorectal adenomatous polyposis.

To uncover novel causative genes in patients with unexplained adenomatous polyposis, a model disease for colorectal cancer, we performed a genome-wide analysis of germline copy number variants (CNV) in a large, well characterized APC and MUTYH mutation negative patient cohort followed by a targeted next generation sequencing (NGS) approach. Genomic DNA from 221 unrelated German patients was genotyped on high-resolution SNP arrays. Putative CNVs were filtered according to stringent criteria, compared with those of 531 population-based German controls, and validated by qPCR. Candidate genes were prioritized using in silico, expression, and segregation analyses, data mining and enrichment analyses of genes and pathways. In 27% of the 221 unrelated patients, a total of 77 protein coding genes displayed rare, nonrecurrent, germline CNVs. The set included 26 candidates with molecular and cellular functions related to tumorigenesis. Targeted high-throughput sequencing found truncating point mutations in 12% (10/77) of the prioritized genes. No clear evidence was found for autosomal recessive subtypes. Six patients had potentially causative mutations in more than one of the 26 genes. Combined with data from recent studies of early-onset colorectal and breast cancer, recurrent potential loss-of-function alterations were detected in CNTN6, FOCAD (KIAA1797), HSPH1, KIF26B, MCM3AP, YBEY and in three genes from the ARHGAP family. In the canonical Wnt pathway oncogene CTNNB1 (ß-catenin), two potential gain-of-function mutations were found. In conclusion, the present study identified a group of rarely affected genes which are likely to predispose to colorectal adenoma formation and confirmed previously published candidates for tumor predisposition as etiologically relevant.

The Cpx envelope stress response regulates and is regulated by small noncoding RNAs.

The Escherichia coli genome encodes approximately 30 two-component systems that are required for sensing and responding to a variety of environmental and physiological cues. Recent studies have revealed numerous regulatory connections between two-component systems and small noncoding RNAs (sRNAs), which posttranscriptionally regulate gene expression by base pairing with target mRNAs. In this study, we investigated the role of sRNAs in the CpxAR two-component system, which detects and mediates an adaptive response to potentially lethal protein misfolding in the Gram-negative bacterial envelope. Here, we showed for the first time that sRNAs are members of the Cpx regulon. We found that CpxR binds to the promoter regions and regulates expression of two sRNA genes, cyaR and rprA. We also investigated the roles that these sRNAs play in the Cpx response. Cpx repression of cyaR expression creates a feed-forward loop, in which CpxAR increases expression of the inner membrane protein YqaE both directly at the transcriptional level and indirectly at the translational level. Moreover, we found that RprA exerts negative feedback on the Cpx response, reducing Cpx activity in a manner that is dependent on the response regulator CpxR but independent of all of RprA's previously described targets. sRNAs therefore permit the fine-tuning of Cpx pathway activity and its regulation of target genes, which could assist bacterial survival in the face of envelope stress.

Gastrointestinal microbiota-mediated control of enteric pathogens.

The gastrointestinal (GI) microbiota is a complex community of microorganisms residing within the mammalian gastrointestinal tract. The GI microbiota is vital to the development of the host immune system and plays a crucial role in human health and disease. The composition of the GI microbiota differs immensely among individuals yet specific shifts in composition and diversity have been linked to inflammatory bowel disease, obesity, atopy, and susceptibility to infection. In this review, we describe the GI microbiota and its role in enteric diseases caused by pathogenic Escherichia coli, Salmonella enterica, and Clostridium difficile. We discuss the central role of the GI microbiota in protective immunity, resistance to enteric pathogens, and resolution of enteric colitis.

Hfq reduces envelope stress by controlling expression of envelope-localized proteins and protein complexes in enteropathogenic Escherichia coli.

Gram-negative bacteria possess several envelope stress responses that detect and respond to damage to this critical cellular compartment. The σ(E) envelope stress response senses the misfolding of outer membrane proteins (OMPs), while the Cpx two-component system is believed to detect the misfolding of periplasmic and inner membrane proteins. Recent studies in several Gram-negative organisms found that deletion of hfq, encoding a small RNA chaperone protein, activates the σ(E) envelope stress response. In this study, we assessed the effects of deleting hfq upon activity of the σ(E) and Cpx responses in non-pathogenic and enteropathogenic (EPEC) strains of Escherichia coli. We found that the σ(E) response was activated in Δhfq mutants of all E. coli strains tested, resulting from the misregulation of OMPs. The Cpx response was activated by loss of hfq in EPEC, but not in E. coli K-12. Cpx pathway activation resulted in part from overexpression of the bundle-forming pilus (BFP) in EPEC Δhfq. We found that Hfq repressed expression of the BFP via PerA, a master regulator of virulence in EPEC. This study shows that Hfq has a more extensive role in regulating the expression of envelope proteins and horizontally acquired virulence genes in E. coli than previously recognized.

Spectrum of novel mutations found in Waardenburg syndrome types 1 and 2: implications for molecular genetic diagnostics.

OBJECTIVES: Till date, mutations in the genes PAX3 and MITF have been described in Waardenburg syndrome (WS), which is clinically characterised by congenital hearing loss and pigmentation anomalies. Our study intended to determine the frequency of mutations and deletions in these genes, to assess the clinical phenotype in detail and to identify rational priorities for molecular genetic diagnostics procedures. DESIGN: Prospective analysis. PATIENTS: 19 Caucasian patients with typical features of WS underwent stepwise investigation of PAX3 and MITF. When point mutations and small insertions/deletions were excluded by direct sequencing, copy number analysis by multiplex ligation-dependent probe amplification was performed to detect larger deletions and duplications. Clinical data and photographs were collected to facilitate genotype-phenotype analyses. SETTING: All analyses were performed in a large German laboratory specialised in genetic diagnostics. RESULTS: 15 novel and 4 previously published heterozygous mutations in PAX3 and MITF were identified. Of these, six were large deletions or duplications that were only detectable by copy number analysis. All patients with PAX3 mutations had typical phenotype of WS with dystopia canthorum (WS1), whereas patients with MITF gene mutations presented without dystopia canthorum (WS2). In addition, one patient with bilateral hearing loss and blue eyes with iris stroma dysplasia had a de novo missense mutation (p.Arg217Ile) in MITF. MITF 3-bp deletions at amino acid position 217 have previously been described in patients with Tietz syndrome (TS), a clinical entity with hearing loss and generalised hypopigmentation. CONCLUSIONS: On the basis of these findings, we conclude that sequencing and copy number analysis of both PAX3 and MITF have to be recommended in the routine molecular diagnostic setting for patients, WS1 and WS2. Furthermore, our genotype-phenotype analyses indicate that WS2 and TS correspond to a clinical spectrum that is influenced by MITF mutation type and position.

Using reporter genes and the Escherichia coli ASKA overexpression library in screens for regulators of the Gram negative envelope stress response.

We describe methods for screening the E. coliASKA overexpression library for clones that lead to altered expression of reporter genes. First, a promoter of interest is cloned upstream of either the lacZor luxCDABEgenes to yield reporter genes in which transcription is proportional to the levels of ß-galactosidase or luminescence produced by strains carrying the reporter. The ASKA library is then condensed into two 96-well plates resulting in mixed preparations of 12 plasmids in each well. The plasmids in each well are transformed into the reporter strain and transformants are screened for either altered ß-galactosidase or light production. The genes contained in ASKA clones that result in altered reporter gene expression are amplified and sequenced and the ASKA clone for the gene identified is retransformed into the parent reporter strain to confirm the effect. We have used screens like this one to look for new E. coligenes that, when over-expressed, result in the altered expression of promoters that are regulated by the envelope stress response. The identity of the clones can yield information about the nature of inducing cues and/or additional regulatory molecules. The techniques are broadly applicable to any microbial function of interest.

Deep intronic APC mutations explain a substantial proportion of patients with familial or early-onset adenomatous polyposis.

To uncover pathogenic deep intronic variants in patients with colorectal adenomatous polyposis, in whom no germline mutation in the APC or MUTYH genes can be identified by routine diagnostics, we performed a systematic APC messenger RNA analysis in 125 unrelated mutation-negative cases. Overall, we identified aberrant transcripts in 8% of the patients (familial cases 30%; early-onset manifestation 21%). In eight of them, two different out-of-frame pseudoexons were found consisting of a 167-bp insertion from intron 4 in five families with a shared founder haplotype and a 83-bp insertion from intron 10 in three patients. The pseudoexon formation was caused by three different heterozygous germline mutations, which are supposed to activate cryptic splice sites. In conclusion, a few deep intronic mutations contribute substantially to the APC mutation spectrum. Complementary DNA analysis and/or target sequencing of intronic regions should be considered as an additional mutation discovery approach in polyposis patients.