RESUMEN
HOXB13 is a key lineage homeobox transcription factor that plays a critical role in the differentiation of the prostate gland. Several studies have suggested that HOXB13 alterations may be involved in prostate cancer development and progression. Despite its potential biological relevance, little is known about the expression of HOXB13 across the disease spectrum of prostate cancer. To this end, we validated a HOXB13 antibody using genetic controls and investigated HOXB13 protein expression in murine and human developing prostates, localized prostate cancers, and metastatic castration-resistant prostate cancers. We observed that HOXB13 expression increases during later stages of murine prostate development. All localized prostate cancers showed HOXB13 protein expression. Interestingly, lower HOXB13 expression levels were observed in higher-grade tumors, although no significant association between HOXB13 expression and recurrence or disease-specific survival was found. In advanced metastatic prostate cancers, HOXB13 expression was retained in the majority of tumors. While we observed lower levels of HOXB13 protein and mRNA levels in tumors with evidence of lineage plasticity, 84% of androgen receptor-negative castration-resistant prostate cancers and neuroendocrine prostate cancers (NEPCs) retained detectable levels of HOXB13. Notably, the reduced expression observed in NEPCs was associated with a gain of HOXB13 gene body CpG methylation. In comparison to the commonly used prostate lineage marker NKX3.1, HOXB13 showed greater sensitivity in detecting advanced metastatic prostate cancers. Additionally, in a cohort of 837 patients, 383 with prostatic and 454 with non-prostatic tumors, we found that HOXB13 immunohistochemistry had a 97% sensitivity and 99% specificity for prostatic origin. Taken together, our studies provide valuable insight into the expression pattern of HOXB13 during prostate development and cancer progression. Furthermore, our findings support the utility of HOXB13 as a diagnostic biomarker for prostate cancer, particularly to confirm the prostatic origin of advanced metastatic castration-resistant tumors. © 2023 The Pathological Society of Great Britain and Ireland.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Neoplasias de la Próstata , Animales , Humanos , Masculino , Ratones , Genes Homeobox , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Próstata/patología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Reino UnidoRESUMEN
The mechanisms by which bacterial cells generate helical cell shape and its functional role are poorly understood. Helical shape of the human pathogen Helicobacter pylori may facilitate penetration of the thick gastric mucus where it replicates. We identified four genes required for helical shape: three LytM peptidoglycan endopeptidase homologs (csd1-3) and a ccmA homolog. Surrounding the cytoplasmic membrane of most bacteria, the peptidoglycan (murein) sacculus is a meshwork of glycan strands joined by peptide crosslinks. Intact cells and isolated sacculi from mutants lacking any single csd gene or ccmA formed curved rods and showed increased peptidoglycan crosslinking. Quantitative morphological analyses of multiple-gene deletion mutants revealed each protein uniquely contributes to a shape-generating pathway. This pathway is required for robust colonization of the stomach in spite of normal directional motility. Our findings suggest that the coordinated action of multiple proteins relaxes peptidoglycan crosslinking, enabling helical cell curvature and twist.
Asunto(s)
Infecciones por Helicobacter/microbiología , Helicobacter pylori/citología , Helicobacter pylori/patogenicidad , Peptidoglicano/metabolismo , Estómago/microbiología , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Endopeptidasas/metabolismo , Femenino , Helicobacter pylori/enzimología , Helicobacter pylori/genética , Metaloexopeptidasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Organismos Libres de Patógenos EspecíficosRESUMEN
Helical cell shape appears throughout the bacterial phylogenetic tree. Recent exciting work characterizing cell shape mutants in a number of curved and helical Proteobacteria is beginning to suggest possible mechanisms and provide tools to assess functional significance. We focus here on Caulobacter crescentus, Vibrio cholerae, Helicobacter pylori, and Campylobacter jejuni, organisms from three classes of Proteobacteria that live in diverse environments, from freshwater and saltwater to distinct compartments within the gastrointestinal tract of humans and birds. Comparisons among these bacteria reveal common themes as well as unique solutions to the task of maintaining cell curvature. While motility appears to be influenced in all these bacteria when cell shape is perturbed, consequences on niche colonization are diverse, suggesting the need to consider additional selective pressures.
Asunto(s)
Morfogénesis , Proteobacteria/citología , Proteobacteria/crecimiento & desarrollo , Adaptación Biológica , Animales , Microbiología Ambiental , HumanosRESUMEN
Helicobacter pylori chronically infects the stomach of approximately half of the world's population. Manifestation of clinical diseases associated with H. pylori infection, including cancer, is driven by strain properties and host responses; and as chronic infection persists, both are subject to change. Previous studies have documented frequent and extensive within-host bacterial genetic variation. To define how within-host diversity contributes to phenotypes related to H. pylori pathogenesis, this project leverages a collection of 39 clinical isolates acquired prospectively from a single subject at two time points and from multiple gastric sites. During the six years separating collection of these isolates, this individual, initially harboring a duodenal ulcer, progressed to gastric atrophy and concomitant loss of acid secretion. Whole genome sequence analysis identified 1,767 unique single nucleotide polymorphisms (SNPs) across isolates and a nucleotide substitution rate of 1.3x10-4 substitutions/site/year. Gene ontology analysis identified cell envelope genes among the genes with excess accumulation of nonsynonymous SNPs (nSNPs). A maximum likelihood tree based on genetic similarity clusters isolates from each time point separately. Within time points, there is segregation of subgroups with phenotypic differences in bacterial morphology, ability to induce inflammatory cytokines, and mouse colonization. Higher inflammatory cytokine induction in recent isolates maps to shared polymorphisms in the Cag PAI protein, CagY, while rod morphology in a subgroup of recent isolates mapped to eight mutations in three distinct helical cell shape determining (csd) genes. The presence of subgroups with unique genetic and phenotypic properties suggest complex selective forces and multiple niches within the stomach during chronic infection.
Asunto(s)
Úlcera Duodenal/microbiología , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/genética , Helicobacter pylori/genética , Gastropatías/microbiología , Animales , Atrofia/microbiología , Enfermedad Crónica , Ácido Gástrico , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Fenotipo , Polimorfismo de Nucleótido Simple , Gastropatías/patologíaRESUMEN
Gram-negative bacteria are surrounded by two membrane bilayers separated by a space termed the periplasm. The periplasm is a multipurpose compartment separate from the cytoplasm whose distinct reducing environment allows more efficient and diverse mechanisms of protein oxidation, folding, and quality control. The periplasm also contains structural elements and important environmental sensing modules, and it allows complex nanomachines to span the cell envelope. Recent work indicates that the size or intermembrane distance of the periplasm is controlled by periplasmic lipoproteins that anchor the outer membrane to the periplasmic peptidoglycan polymer. This periplasm intermembrane distance is critical for sensing outer membrane damage and dictates length of the flagellar periplasmic rotor, which controls motility. These exciting results resolve longstanding debates about whether the periplasmic distance has a biological function and raise the possibility that the mechanisms for maintenance of periplasmic size could be exploited for antibiotic development.
Asunto(s)
Bacterias Gramnegativas/fisiología , Periplasma/metabolismo , Periplasma/fisiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Bacterianas/fisiología , Membrana Celular/fisiología , Pared Celular/fisiología , Citoplasma , Peptidoglicano/metabolismo , Análisis EspacialRESUMEN
Evident in its name, the gastric pathogen Helicobacter pylori has a helical cell morphology which facilitates efficient colonization of the human stomach. An improved light-focusing strategy allowed us to robustly distinguish even subtle perturbations of H. pylori cell morphology by deviations in light-scattering properties measured by flow cytometry. Profiling of an arrayed genome-wide deletion library identified 28 genes that influence different aspects of cell shape, including properties of the helix, cell length or width, cell filament formation, cell shape heterogeneity, and cell branching. Included in this mutant collection were two that failed to form any helical cells, a soluble lytic transglycosylase and a previously uncharacterized putative multipass inner membrane protein HPG27_0728, renamed Csd7. A combination of cell fractionation, mutational, and immunoprecipitation experiments show that Csd7 and Csd2 collaborate to stabilize the Csd1 peptidoglycan (PG) endopeptidase. Thus, both csd2 and csd7 mutants show the same enhancement of PG tetra-pentapeptide cross-linking as csd1 mutants. Csd7 also links Csd1 with the bactofilin CcmA via protein-protein interactions. Although Csd1 is stable in ccmA mutants, these mutants show altered PG tetra-pentapeptide cross-linking, suggesting that Csd7 may directly or indirectly activate as well as stabilize Csd1. These data begin to illuminate a highly orchestrated program to regulate PG modifications that promote helical shape, which includes nine nonessential nonredundant genes required for helical shape and 26 additional genes that further modify H. pylori's cell morphology.IMPORTANCE The stomach ulcer and cancer-causing pathogen Helicobacter pylori has a helical cell shape which facilitates stomach infection. Using light scattering to measure perturbations of cell morphology, we identified 28 genes that influence different aspects of cell shape. A mutant in a previously uncharacterized protein renamed Csd7 failed to form any helical cells. Biochemical analyses showed that Csd7 collaborates with other proteins to stabilize the cell wall-degrading enzyme Csd1. Csd7 also links Csd1 with a putative filament-forming protein via protein-protein interactions. These data suggest that helical cell shape arises from a highly orchestrated program to regulate cell wall modifications. Targeting of this helical cell shape-promoting program could offer new ways to block infectivity of this important human pathogen.
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Membrana Externa Bacteriana/química , Proteínas Bacterianas/química , Endopeptidasas/química , Genoma Bacteriano , Helicobacter pylori/citología , Helicobacter pylori/genética , Proteínas Bacterianas/genética , Pared Celular , Citoesqueleto/química , Endopeptidasas/genética , MutaciónRESUMEN
Half of all humans harbor Helicobacter pylori in their stomachs. Helical cell shape is thought to facilitate H. pylori's ability to bore into the protective mucus layer in a corkscrew-like motion, thereby enhancing colonization of the stomach. H. pylori cell shape mutants show impaired colonization of the mouse stomach, highlighting the importance of cell shape in infection. To gain a deeper understanding of how helical cell morphology promotes host colonization by H. pylori, we used three-dimensional confocal microscopy to visualize the clinical isolate PMSS1 and an isogenic straight-rod mutant (Δcsd6) within thick longitudinal mouse stomach sections. We also performed volumetric image analysis to quantify the number of bacteria residing within corpus and antral glands in addition to measuring total CFU. We found that straight rods show attenuation during acute colonization of the stomach (1 day or 1 week postinfection) as measured by total CFU. Our quantitative imaging revealed that wild-type bacteria extensively colonized antral glands at 1 week postinfection, while csd6 mutants showed variable colonization of the antrum at this time point. During chronic infection (1 or 3 months postinfection), total CFU were highly variable but similar for wild-type and straight rods. Both wild-type and straight rods persisted and expanded in corpus glands during chronic infection. However, the straight rods showed reduced inflammation and disease progression. Thus, helical cell shape contributes to tissue interactions that promote inflammation during chronic infection, in addition to facilitating niche acquisition during acute infection.
Asunto(s)
Infecciones por Helicobacter/microbiología , Helicobacter pylori/citología , Helicobacter pylori/crecimiento & desarrollo , Estómago/patología , Animales , Adhesión Bacteriana , Enfermedad Crónica , Femenino , Infecciones por Helicobacter/patología , Helicobacter pylori/genética , Humanos , Ratones Endogámicos C57BL , Antro Pilórico/microbiología , Antro Pilórico/patología , Estómago/microbiologíaRESUMEN
Chronic infection with Helicobacter pylori can lead to the development of gastric ulcers and stomach cancers. The helical cell shape of H. pylori promotes stomach colonization. Screens for loss of helical shape have identified several periplasmic peptidoglycan (PG) hydrolases and non-enzymatic putative scaffolding proteins, including Csd5. Both over and under expression of the PG hydrolases perturb helical shape, but the mechanism used to coordinate and localize their enzymatic activities is not known. Using immunoprecipitation and mass spectrometry we identified Csd5 interactions with cytosolic proteins CcmA, a bactofilin required for helical shape, and MurF, a PG precursor synthase, as well as the inner membrane spanning ATP synthase. A combination of Csd5 domain deletions, point mutations, and transmembrane domain chimeras revealed that the N-terminal transmembrane domain promotes MurF, CcmA, and ATP synthase interactions, while the C-terminal SH3 domain mediates PG binding. We conclude that Csd5 promotes helical shape as part of a membrane associated, multi-protein shape complex that includes interactions with the periplasmic cell wall, a PG precursor synthesis enzyme, the bacterial cytoskeleton, and ATP synthase.
Asunto(s)
Pared Celular/metabolismo , Citoesqueleto/metabolismo , Helicobacter pylori/citología , Helicobacter pylori/enzimología , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Péptido Sintasas/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Eliminación de Gen , Helicobacter pylori/genética , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/genética , Péptido Sintasas/química , Péptido Sintasas/genética , Periplasma/metabolismo , Análisis de Secuencia de ProteínaRESUMEN
Chronic infection with Helicobacter pylori causes peptic ulcers and stomach cancer in a subset of infected individuals. While standard eradication therapy includes multiple antibiotics, treatment failure due to resistance is an increasing clinical problem. Accurate assessment of H. pylori antimicrobial resistance has been limited by slow growth and sampling of few isolates per subject. We established a method to simultaneously quantify H. pylori clarithromycin-resistant (mutant) and -susceptible (wild-type) 23S rRNA gene alleles in both stomach and stool samples using droplet digital PCR (ddPCR). In 49 subjects, we assessed the performance of these assays alongside clarithromycin MIC testing of up to 16 H. pylori isolates per subject and included both cancer (25 subjects) and noncancer (24 subjects) cases. Gastric ddPCR and H. pylori culture showed agreement with urea breath test (UBT) detection of infection in 94% and 88% of subjects, respectively, while stool ddPCR showed agreement with UBT in 92% of subjects. Based on MIC testing of 43 culture-positive cases, 20 subjects had only susceptible isolates, 14 had a mix of susceptible and resistant isolates, and 9 had only resistant isolates. ddPCR of gastric samples indicated that 21 subjects had only wild-type alleles, 13 had a mixed genotype, and 9 had only mutant alleles. Stool ddPCR detected mutant alleles in four subjects for which mutant alleles were not detected by stomach ddPCR, and no resistant isolates were cultured. Our results indicate that ddPCR detects H. pylori clarithromycin resistance-associated genotypes, especially in the context of heteroresistance.
Asunto(s)
Antibacterianos/farmacología , Claritromicina/farmacología , Farmacorresistencia Bacteriana/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Adulto , Anciano , Farmacorresistencia Bacteriana/efectos de los fármacos , Heces/microbiología , Femenino , Mucosa Gástrica/microbiología , Variación Genética , Genotipo , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/genética , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , ARN Ribosómico 23S/genéticaRESUMEN
In this review, we highlight progress in the last year in characterizing known virulence factors like flagella and the Cag type IV secretion system with sophisticated structural and biochemical approaches to yield new insight on the assembly and functions of these critical virulence determinants. Several aspects of Helicobacter pylori physiology were newly explored this year and evaluated for their functions during stomach colonization, including a fascinating role for the essential protease HtrA in allowing access of H. pylori to the basolateral side of the gastric epithelium through cleavage of the tight junction protein E-cadherin to facilitate CagA delivery. Molecular biology tools standard in model bacteria, including regulated gene expression during animal infection and fluorescent reporter gene fusions, were newly applied to H. pylori to explore functions for urease beyond initial colonization and establish high salt consumption as a mediator of gene expression changes. New sequencing technologies enabled validation of long postulated roles for DNA methylation in regulating H. pylori gene expression. On the cell biology side, elegant work using lineage tracing in the murine model and organoid primary cell culture systems has provided new insights into how H. pylori manipulates gastric tissue functions, locally and at a distance, to promote its survival in the stomach and induce pathologic changes. Finally, new work has bolstered the case for genomic variation as an important mechanism to generate phenotypic diversity during changing environmental conditions in the context of diet manipulation in animal infection models and during human experimental infection after vaccination.
Asunto(s)
Infecciones por Helicobacter/patología , Animales , Mucosa Gástrica/microbiología , Genoma Bacteriano/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/inmunología , Helicobacter pylori/patogenicidad , Humanos , Virulencia , Factores de VirulenciaRESUMEN
BACKGROUND: Treatment of Helicobacter pylori infection is often empiric; however, current guidelines for management of Helicobacter pylori infection advise against the use of standard triple therapy (clarithromycin, amoxicillin, and proton-pump inhibitor) when clarithromycin resistance exceeds 20%. We developed and tested a new culture-free assay to detect clarithromycin resistance-conferring mutations to determine the prevalence of H. pylori clarithromycin resistance in patients from the United States Pacific Northwest. MATERIALS AND METHODS: Droplet digital PCR (ddPCR) was used to detect the H. pylori 23S rRNA gene, and resistance-conferring mutations, in archived, formalin-fixed, paraffin-embedded (FFPE) gastric tissue and to retrospectively determine the prevalence of clarithromycin-resistant H. pylori among 110 patients at an academic medical center in the Northwest United States between 2012 and 2014. RESULTS: Of 102 patients with the H. pylori 23S rRNA gene detected by the ddPCR assay, 45 (44%) had clarithromycin resistance mutations. Thirty-three of the 45 patients with clarithromycin resistance mutations had a mix of wild-type and resistance alleles. Prevalence of clarithromycin resistance mutations differed among racial groups and was highest among Asians, with mutations detected in 14 (67%) of the 21 patient samples. CONCLUSIONS: The prevalence of clarithromycin resistance detected in this region exceeds 20%, indicating that standard triple therapy should not be the first-line antibiotic treatment for H. pylori infection. Culture-free assays for detecting clarithromycin resistance mutations can be performed on archived tissue samples and will aid in informing tailored treatment for effective H. pylori eradication.
Asunto(s)
Claritromicina/farmacología , Farmacorresistencia Bacteriana/genética , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/genética , Humanos , Mutación/genética , Reacción en Cadena de la Polimerasa , Prevalencia , Estudios RetrospectivosRESUMEN
The helical shape of the human stomach pathogen Helicobacter pylori has been suggested to provide mechanical advantage for penetrating the viscous stomach mucus layer. Using single-cell tracking and quantitative morphology analysis, we document marked variation in cell body helical parameters and flagellum number among H. pylori strains leading to distinct and broad speed distributions in broth and viscous gastric mucin media. These distributions reflect both temporal variation in swimming speed and morphologic variation within the population. Isogenic mutants with straight-rod morphology showed 7-21% reduction in speed and a lower fraction of motile bacteria. Mutational perturbation of flagellum number revealed a 19% increase in speed with 4 versus 3 median flagellum number. Resistive force theory modeling incorporating variation of both cell shape and flagellum number predicts qualitative speed differences of 10-30% among strains. However, quantitative comparisons suggest resistive force theory underestimates the influence of cell body shape on speed for helical shaped bacteria.
Asunto(s)
Adaptación Fisiológica , Flagelos/fisiología , Helicobacter pylori/fisiología , Locomoción , Rastreo Celular , Medios de Cultivo/química , Humanos , Mucinas/metabolismo , Análisis de la Célula IndividualRESUMEN
Peptidoglycan modifying carboxypeptidases (CPs) are important determinants of bacterial cell shape. Here, we report crystal structures of Csd4, a three-domain protein from the human gastric pathogen Helicobacter pylori. The catalytic zinc in Csd4 is coordinated by a rare His-Glu-Gln configuration that is conserved among most Csd4 homologs, which form a distinct subfamily of CPs. Substitution of the glutamine to histidine, the residue found in prototypical zinc carboxypeptidases, resulted in decreased enzyme activity and inhibition by phosphate. Expression of the histidine variant at the native locus in a H. pylori csd4 deletion strain did not restore the wild-type helical morphology. Biochemical assays show that Csd4 can cleave a tripeptide peptidoglycan substrate analog to release m-DAP. Structures of Csd4 with this substrate analog or product bound at the active site reveal determinants of peptidoglycan specificity and the mechanism to cleave an isopeptide bond to release m-DAP. Our data suggest that Csd4 is the archetype of a new CP subfamily with a domain scheme that differs from this large family of peptide-cleaving enzymes.
Asunto(s)
Proteínas Bacterianas/química , Carboxipeptidasas/química , Glutamina/metabolismo , Helicobacter pylori/enzimología , Zinc/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Carboxipeptidasas/genética , Carboxipeptidasas/metabolismo , Glutamina/química , Glutamina/genética , Helicobacter pylori/citología , Ligandos , Datos de Secuencia Molecular , Mutación , Péptidos/metabolismo , Peptidoglicano/metabolismo , Unión ProteicaRESUMEN
All organisms have pathways that repair the genome, ensuring their survival and that of their progeny. But these pathways also serve to diversify the genome, causing changes at the nucleotide, whole gene, and genome structure levels. Sequencing of bacteria has revealed wide allelic diversity and differences in gene content within the same species, highlighting the importance of understanding pathways of recombination and DNA repair. The human stomach pathogen Helicobacter pylori is an excellent model system for studying these pathways. H. pylori harbors major recombination and repair pathways and is naturally competent, facilitating its ability to diversify its genome. Elucidation of DNA recombination, repair, and diversification programs in this pathogen will reveal connections between these pathways and their importance to infection.
Asunto(s)
Reparación del ADN , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Recombinación Genética , Animales , Helicobacter pylori/fisiología , HumanosRESUMEN
BACKGROUND: Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning. MATERIALS AND METHODS: Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital. RESULTS: The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool. CONCLUSIONS: The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen.
Asunto(s)
Antígenos Bacterianos/análisis , Carga Bacteriana/métodos , Proteínas Bacterianas/análisis , Heces/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Factores de Virulencia/análisis , Adolescente , Adulto , Anciano , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Niño , Preescolar , Costa Rica , Femenino , Técnicas de Genotipaje/métodos , Helicobacter pylori/genética , Humanos , Lactante , Masculino , Persona de Mediana Edad , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad , Estados Unidos , Factores de Virulencia/genética , Adulto JovenRESUMEN
Helicobacter pylori from different individuals exhibits substantial genetic diversity. However, the kinetics of bacterial diversification after infection with a single strain is poorly understood. We investigated evolution of H. pylori following long-term infection in the primate stomach; Rhesus macaques were infected with H. pylori strain USU101 and then followed for 10 years. H. pylori was regularly cultured from biopsies, and single colony isolates were analyzed. At 1-year, DNA fingerprinting showed that all output isolates were identical to the input strain; however, at 5-years, different H. pylori fingerprints were observed. Microarray-based comparative genomic hybridization revealed that long term persistence of USU101 in the macaque stomach was associated with specific whole gene changes. Further detailed investigation showed that levels of the BabA protein were dramatically reduced within weeks of infection. The molecular mechanisms behind this reduction were shown to include phase variation and gene loss via intragenomic rearrangement, suggesting strong selective pressure against BabA expression in the macaque model. Notably, although there is apparently strong selective pressure against babA, babA is required for establishment of infection in this model as a strain in which babA was deleted was unable to colonize experimentally infected macaques.
Asunto(s)
Variación Genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/aislamiento & purificación , Adhesinas Bacterianas/genética , Animales , Biopsia , Hibridación Genómica Comparativa , Dermatoglifia del ADN , ADN Bacteriano/genética , Modelos Animales de Enfermedad , Reordenamiento Génico , Estudios Longitudinales , Macaca mulatta , Análisis por Micromatrices , Selección Genética , Estómago/microbiologíaRESUMEN
The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi-faceted peptidoglycan (PG) modification programme involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape-determining genes, we employed flow cytometry with fluorescence-activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37-fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has ld-carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27_782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori.
Asunto(s)
Proteínas Bacterianas/metabolismo , Genes Bacterianos , Helicobacter pylori/citología , Helicobacter pylori/genética , Proteínas Bacterianas/genética , Evolución Biológica , Carboxipeptidasas/genética , Carboxipeptidasas/metabolismo , Movimiento Celular , Pared Celular/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Citometría de Flujo , Helicobacter pylori/enzimología , Mutación , Peptidoglicano/metabolismoRESUMEN
Helical cell shape of the gastric pathogen Helicobacter pylori has been suggested to promote virulence through viscosity-dependent enhancement of swimming velocity. However, H. pylori csd1 mutants, which are curved but lack helical twist, show normal velocity in viscous polymer solutions and the reason for their deficiency in stomach colonization has remained unclear. Characterization of new rod shaped mutants identified Csd4, a DL-carboxypeptidase of peptidoglycan (PG) tripeptide monomers and Csd5, a putative scaffolding protein. Morphological and biochemical studies indicated Csd4 tripeptide cleavage and Csd1 crosslinking relaxation modify the PG sacculus through independent networks that coordinately generate helical shape. csd4 mutants show attenuation of stomach colonization, but no change in proinflammatory cytokine induction, despite four-fold higher levels of Nod1-agonist tripeptides in the PG sacculus. Motility analysis of similarly shaped mutants bearing distinct alterations in PG modifications revealed deficits associated with shape, but only in gel-like media and not viscous solutions. As gastric mucus displays viscoelastic gel-like properties, our results suggest enhanced penetration of the mucus barrier underlies the fitness advantage conferred by H. pylori's characteristic shape.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori , Peptidoglicano/metabolismo , Animales , Fenómenos Fisiológicos Bacterianos/genética , Pared Celular/genética , Modelos Animales de Enfermedad , Femenino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/citología , Helicobacter pylori/patogenicidad , Helicobacter pylori/fisiología , Ratones , Ratones Endogámicos C57BL , Movimiento , Moco/metabolismo , Moco/microbiología , MutaciónRESUMEN
Helicobacter pylori is a bacterial pathogen that colonizes the gastric niche of â¼ 50% of the human population worldwide and is known to cause peptic ulceration and gastric cancer. Pathology of infection strongly depends on a cag pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). Here, we aimed to identify as yet unknown bacterial factors involved in cagPAI effector function and performed a large-scale screen of an H. pylori transposon mutant library using activation of the pro-inflammatory transcription factor NF-κB in human gastric epithelial cells as a measure of T4SS function. Analysis of â¼ 3000 H. pylori mutants revealed three non-cagPAI genes that affected NF-κB nuclear translocation. Of these, the outer membrane protein HopQ from H. pylori strain P12 was essential for CagA translocation and for CagA-mediated host cell responses such as formation of the hummingbird phenotype and cell scattering. Besides that, deletion of hopQ reduced T4SS-dependent activation of NF-κB, induction of MAPK signalling and secretion of interleukin 8 (IL-8) in the host cells, but did not affect motility or the quantity of bacteria attached to host cells. Hence, we identified HopQ as a non-cagPAI-encoded cofactor of T4SS function.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Sistemas de Secreción Bacterianos , Helicobacter pylori/metabolismo , Factores de Virulencia/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Línea Celular , Elementos Transponibles de ADN , Células Epiteliales/microbiología , Eliminación de Gen , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Humanos , Interleucina-8/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , FN-kappa B/metabolismo , Análisis de Secuencia de ADN , Transducción de Señal , Factores de Virulencia/genéticaRESUMEN
Background: American Indian and Alaska Native people in the United States experience high rates of stomach cancer. Helicobacter pylori infection is a significant risk factor for stomach cancer, and H. pylori strains that carry the cagA gene are linked to greater gastrointestinal disease severity. Yet, little is known about H. pylori and cagA infections in American Indian and Alaska Native people, particularly at the tribal level. We assessed the prevalence and risk factors of H. pylori infection and cagA gene carriage in tribal members from the Navajo Nation. Materials and Methods: We conducted a cross-sectional study with adults from the Navajo Nation. Stool samples collected from participants were analyzed with droplet digital PCR for H. pylori 16S ribosomal and cagA virulence genes. Self-administered health and food questionnaires were mailed to participants to collect information on sociodemographic, health, lifestyle, and environmental risk factors for H. pylori infection. Logistic regression assessed the association between risk factors and H. pylori infection and cagA gene carriage. Results: Among 99 adults, the median age was 45 (age range: 18 to 79 years), and 73.7% were female. About 56.6% (95% CI: 46.2-66.5) of participants were infected with H. pylori. Of H. pylori-infected participants, 78.6% (95% CI: 65.6-88.4) were cagA-gene positive. No significant associations of relevant risk factors with H. pylori and cagA-gene positive infections were noted. Conclusions: In a community-based study population, a substantial proportion of adult tribal members had H. pylori and cagA-gene positive infections. Given these high proportions, culturally appropriate prevention strategies and interventions addressing H. pylori infections present an avenue for additional research and stomach cancer prevention in the Navajo Nation.