Ferrous and ferric iron acquisition in Campylobacter jejuni

Scope and Method of study: Identification of colonization and virulence determinants that contribute to C. jejuni infection and survival in the gastrointestinal tract of the animal host would be an important tool in designing intervention strategies to combat the infection. Availability of iron in the host is a key environmental factor for controlling the establishment of many bacterial infections. The global aim of this project was to decipher the molecular mechanisms of iron acquisition in C. jejuni. We report the characterization of FeoB mediated ferrous iron and TonB mediated ferric iron acquisition in C. jejuni. We used directed mutagenesis to functionally disrupt the feoB and tonB genes. By performing a series of in vitro experiments such as 55Fe2+ uptake assays, cell culture based assays and microarrays; we have studied the role of FeoB in iron transport, iron accumulation and intracellular survival within human and porcine intestinal epithelial cells. TonB systems were characterized in vitro using growth promotion assays, ferric-enterobactin assays and cell culture. The FeoB and TonB systems were extensively assessed for their role in the in vivo colonization of the gastrointestinal tract of the chicken, rabbit ileal loop and the colostrums-deprived neonatal piglet models.Findings and Conclusions: The feoB mutant was significantly impaired in iron uptake and was demonstrated to have decreased cellular iron accumulation. FeoB was found to play significant role in the intracellular survival of C. jejuni within human and porcine epithelial cells. The feoB mutant was significantly affected in its ability to colonize the gastrointestinal tract of chicks, rabbits and neonatal piglets thus highlighting its importance in the gut colonization. Growth promotion assays and ferric-enterobactin assays revealed that TonBs played specific and redundant roles in acquisition of iron from different sources. TonB3 was found to be specifically required for enterobactin utilization, whereas TonB2 and TonB3 were together required for the uptake of hemin and hemoglobin. All the tonB mutants were significantly affected in their ability to colonize the gastrointestinal tract of neonatal piglets. With the exception of tonb2 mutant, all the other series of single and double tonB mutants were affected in their ability to colonize the chicken ceca. Thus, combining our in vitro and in vivo characterization of the FeoB and TonB systems, it is clear that both the systems contribute significantly in iron transport and gastrointestinal colonization of the animal models

Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs

The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity

Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni

Background: During gut colonization, the enteric pathogen Campylobacter jejuni must surmount the toxic effects of reactive oxygen species produced by its own metabolism, the host immune system, and intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology.Results: The mechanisms of oxidative stress defense in C. jejuni were characterized by transcriptional profiling and phenotypic analysis of wild-type and mutant strains. To define the regulon of the peroxide-sensing regulator, PerR, we constructed an isogenic [delta]perR mutant and compared its transcriptome profile with that of the wild-type strain. Transcriptome profiling identified 104 genes that belonged to the PerR regulon. PerR appears to regulate gene expression in a manner that both depends on and is independent of the presence of iron and/or H2O2. Mutation of perR significantly reduced motility. A phenotypic analysis using the chick colonization model showed that the [delta]perR mutant exhibited attenuated colonization behavior. An analysis of changes in the transcriptome induced by exposure to H2O2, cumene hydroperoxide, or menadione revealed differential expression of genes belonging to a variety of biological pathways, including classical oxidative stress defense systems, heat shock response, DNA repair and metabolism, fatty acid biosynthesis, and multidrug efflux pumps. Mutagenic and phenotypic studies of the superoxide dismutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed a role for these proteins in oxidative stress defense and chick gut colonization.Conclusion: This study reveals an interplay between PerR, Fur, iron metabolism and oxidative stress defense, and highlights the role of these elements in C. jejuni colonization of the chick cecum and/or subsequent survival.Peer reviewedVeterinary Pathobiolog

Development and Evaluation of a Novel Taqman Real-Time PCR Assay for Rapid Detection of Mycoplasma bovis: Comparison of Assay Performance with a Conventional PCR Assay and Another Taqman Real-Time PCR Assay

The objective of this study was to develop and validate a Taqman real-time PCR assay for the detection of Mycoplasma bovis (M. bovis). Unique primers targeting the highly conserved house-keeping gene (uvrC) were designed and the probe sequence was derived from a previously published microarray study. There was 100% agreement in the outcome between our assay and the other two published assays for M. bovis detection. The analytical limit of detection of our assay is 83 copies of the uvrC gene. This assay was validated on a total of 214 bovine clinical specimens that were submitted to the Texas A&amp;M Veterinary Medical Diagnostic Laboratory (TVMDL), Texas, USA. The specificity of the assay was assessed to be 100% since no cross-reactivity occurred with 22 other bacterial and other Mycoplasma species. We conclude that the uvrC gene serves as a good and reliable diagnostic marker for the accurate and rapid detection of M. bovis from a wider variety of specimen matrices

Identification of Campylobacter jejuni Genes Involved in the Response to Acidic pH and Stomach Transit▿

Campylobacter jejuni causes food- and waterborne gastroenteritis, and as such it must survive passage through the stomach in order to reach the gastrointestinal tract. While little is known about how C. jejuni survives transit through the stomach, its low infectious dose suggests it is well equipped to sense and respond to acid shock. In this study, the transcriptional profile of C. jejuni NCTC 11168 was obtained after the organism was exposed to in vitro and in vivo (piglet stomach) acid shock. The observed down-regulation of genes encoding ribosomal proteins likely reflects the need to reshuffle energy toward the expression of components required for survival. Acid shock also caused C. jejuni to up-regulate genes involved in stress responses. These included heat shock genes as well as genes involved in the response to oxidative and nitrosative stress. A role for the chaperone clpB in acid resistance was confirmed in vitro. Some genes showed expression patterns that were markedly different in vivo and in vitro, which likely reflects the complexity of the in vivo environment. For instance, transit through the stomach was characterized by up-regulation of genes that encode products that are involved in the use of nitrite as a terminal electron acceptor and down-regulation of genes that are involved in capsular polysaccharide expression. In conclusion, this study has enabled us to understand how C. jejuni modulates gene expression in response to acid shock in vitro and to correlate this with gene expression profiles of C. jejuni as it transits through the host stomach

Development and Evaluation of a Novel Taqman Real-Time PCR Assay for Rapid Detection of Mycoplasma bovis: Comparison of Assay Performance with a Conventional PCR Assay and Another Taqman Real-Time PCR Assay

The objective of this study was to develop and validate a Taqman real-time PCR assay for the detection of Mycoplasma bovis (M. bovis). Unique primers targeting the highly conserved house-keeping gene (uvrC) were designed and the probe sequence was derived from a previously published microarray study. There was 100% agreement in the outcome between our assay and the other two published assays for M. bovis detection. The analytical limit of detection of our assay is 83 copies of the uvrC gene. This assay was validated on a total of 214 bovine clinical specimens that were submitted to the Texas A&amp;M Veterinary Medical Diagnostic Laboratory (TVMDL), Texas, USA. The specificity of the assay was assessed to be 100% since no cross-reactivity occurred with 22 other bacterial and other Mycoplasma species. We conclude that the uvrC gene serves as a good and reliable diagnostic marker for the accurate and rapid detection of M. bovis from a wider variety of specimen matrices

Major Role for FeoB in Campylobacter jejuni Ferrous Iron Acquisition, Gut Colonization, and Intracellular Survival

To assess the importance of ferrous iron acquisition in Campylobacter physiology and pathogenesis, we disrupted and characterized the Fe(2+) iron transporter, FeoB, in Campylobacter jejuni NCTC 11168, 81-176, and ATCC 43431. The feoB mutant was significantly affected in its ability to transport (55)Fe(2+). It accumulated half the amount of iron than the wild-type strain during growth in an iron-containing medium. The intracellular iron of the feoB mutant was localized in the periplasmic space versus the cytoplasm for the wild-type strain. These results indicate that the feoB gene of C. jejuni encodes a functional ferrous iron transport system. Reverse transcriptase PCR analysis revealed the cotranscription of feoB and Cj1397, which encodes a homolog of Escherichia coli feoA. C. jejuni 81-176 feoB mutants exhibited reduced ability to persist in human INT-407 embryonic intestinal cells and porcine IPEC-1 small intestinal epithelial cells compared to the wild type. C. jejuni NCTC 11168 feoB mutant was outcompeted by the wild type for colonization and/or survival in the rabbit ileal loop. The feoB mutants of the three C. jejuni strains were significantly affected in their ability to colonize the chick cecum. And finally, the three feoB mutants were outcompeted by their respective wild-type strains for infection of the intestinal tracts of colostrum-deprived piglets. Taken together, these results demonstrate that FeoB-mediated ferrous iron acquisition contributes significantly to colonization of the gastrointestinal tract during both commensal and infectious relationship, and thus it plays an important role in Campylobacter pathogenesis

Campylobacter jejuni ferric– enterobactin receptor CfrA is TonB3 dependent and mediates iron acquisition from structurally different catechol siderophores

Campylobacter jejuni NCTC11168 does not produce any endogenous siderophores of its own yet requires the CfrA enterobactin transporter for in vivo colonization. In addition, the genome of C. jejuni NCTC11168 contains three distinct TonB energy transduction systems, named TonB1, TonB2, and TonB3, that have not been tested for their role in siderophore uptake or their functional redundancy. We demonstrate that C. jejuni NCTC11168 transports ferric-enterobactin in an energy dependent manner that requires TonB3 for full activity with TonB1 showing partial functional redundancy. Moreover C. jejuni NCTC11168 can utilize a wide variety of structurally different catechol siderophores as sole iron sources during growth. This growth is solely dependent on the CfrA enterobactin transporter and highlights the wide range of substrates that this transporter can recognize. TonB3 is also required for growth on most catechol siderophores. Furthermore, either TonB1 or TonB3 is sufficient for growth on hemin or hemoglobin as a sole iron source demonstrating functional redundancy between TonB1 and TonB3. In vivo colonization assays with isogenic deletion mutants revealed that both TonB1 and TonB3 are required for chick colonization with TonB2 dispensable in this model. These results further highlight the importance of iron transport for efficient C. jejuni colonization

Use of Genome-Wide Expression Profiling and Mutagenesis To Study the Intestinal Lifestyle of Campylobacter jejuni

Campylobacter jejuni is the most common bacterial cause of diarrhea worldwide. To colonize the gut and cause infection, C. jejuni must successfully compete with endogenous microbes for nutrients, resist host defenses, persist in the intestine, and ultimately infect the host. These challenges require the expression of a battery of colonization and virulence determinants. In this study, the intestinal lifestyle of C. jejuni was studied using whole-genome microarray, mutagenesis, and a rabbit ileal loop model. Genes associated with a wide range of metabolic, morphological, and pathological processes were expressed in vivo. The in vivo transcriptome of C. jejuni reflected its oxygen-limited, nutrient-poor, and hyperosmotic environment. Strikingly, the expression of several C. jejuni genes was found to be highly variable between individual rabbits. In particular, differential gene expression suggested that C. jejuni extensively remodels its envelope in vivo by differentially expressing its membrane proteins and by modifying its peptidoglycan and glycosylation composition. Furthermore, mutational analysis of seven genes, hspR, hrcA, spoT, Cj0571, Cj0178, Cj0341, and fliD, revealed an important role for the stringent and heat shock response in gut colonization. Overall, this study provides new insights on the mechanisms of gut colonization, as well as possible strategies employed by Campylobacter to resist or evade the host immune responses