Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni

CosR (Campylobacter oxidative stress regulator; Cj0355c) is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a leading foodborne pathogen causing human gastroenteritis worldwide. Despite importance, the function of CosR remains completely unknown mainly because of cell death caused by its knockout mutation. To overcome this technical limitation, in this study, antisense technology was used to investigate the regulatory function of CosR by modulating the level of CosR expression. Two-dimensional gel electrophoresis (2DGE) was performed to identify the CosR regulon either by suppressing CosR expression with antisense peptide nucleic acid (PNA) or by overexpressing CosR in C. jejuni. According to the results of 2DGE, CosR regulated 32 proteins involved in various cellular processes. Notably, CosR negatively regulated a few key proteins of the oxidative stress response of C. jejuni, such as SodB, Dps, Rrc and LuxS, whereas CosR positively controlled AhpC. Electrophoretic mobility shift assay showed that CosR directly bound to the promoter region of the oxidative stress genes. DNase I footprinting assays identified 21-bp CosR binding sequences in the sodB and ahpC promoters, suggesting CosR specifically recognizes and binds to the regulated genes. Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide. Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type. Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.     

Modulating cell culture oxidative stress reduces protein glycation and acidic charge variant formation

Controlling acidic charge variants is critical for an industrial bioprocess due to the potential impact on therapeutic efficacy and safety. Achieving a consistent charge variant profile at manufacturing scale remains challenging and may require substantial resources to investigate effective control strategies. This is partially due to incomplete understanding of the underlying causes for charge variant formation during the cell culture process. To address this gap, we examined the effects of four process input factors (temperature, iron concentration, feed media age, and antioxidant (rosmarinic acid) concentration) on charge variant profile. These factors were found to affect the charge profile by modulating the cell culture oxidative state. Process conditions with higher acidic peaks corresponded to elevated supernatant peroxide concentration, intracellular reactive oxygen species (ROS) levels, or both. Changes in glycation level were the primary cause of the charge heterogeneity, and for the first time, supernatant peroxide was found to positively correlate with glycation levels. Based on these findings, a novel mathematical model was developed to demonstrate that the rate of acidic species formation was exponentially proportional to the concentrations of supernatant peroxide and protein product. This work provides critical insights into charge variant formation during the cell culture process and highlights the importance of modulating of cell culture oxidative stress for charge variant control.     

Morphological changes of Campylobacter jejuni and Campylobacter coli under various culture conditions and the accompanied changes in the cell composition]

The morphological transformation from the spiral form to the coccoidal form Campylobacter jejuni and Campylobacter coli was studied under various conditions by such techniques as electron microscopy, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and chemical analyses. The conversion from the spiral form to the coccoidal form of Campylobacter in microaerophilic cultivation occurred in about 50% of the cells in 48 h and in about 90% of the cells in 72 h. At higher temperatures (37 C and 42 C) under aerophilic conditions, the spiral-form cells converted easily to the coccoidal form in phosphate-buffered saline. Electron-microscopic studies revealed that the envelopes of the coccoidal-form cells were soft in comparison with those of the spiral-form cells. The LPS and protein contents of the cells reached the highest levels after cultivation for 48 h under microaerophilic condition. The 33 K and 28 K polypeptide contents of 24-h and 48-h cultures were higher than those of 72-h and 96-h cultures.     

Deletion of peb4 gene impairs cell adhesion and biofilm formation in Campylobacter jejuni

Campylobacter jejuni is a microaerophilic bacterium that causes diarrhea in humans. The first step in establishing an infection is adherence to a host cell, which involves two major cell-binding proteins, Peb1A (CBF1) and Peb4 (CBF2). Because the functional role of Peb4 on the cell adhesion remains unclear compared with that of Peb1A, a C. jejuni peb4 deletion mutant was constructed and cell adherence and ability to colonize mouse intestine were studied. The result showed that adherence of the peb4 mutant strain to INT407 cells was 1-2% that of the wild-type strain. Mouse challenge experiments showed a reduced level and duration of intestinal colonization by the mutant compared with the wild-type strain. In addition, fewer peb4 mutant cells than wild-type cells responded to stress by forming a biofilm. Proteomic analysis revealed that the expression levels of proteins involved in various adhesion, transport, and motility functions, which are required for biofilm formation by the pathogen, were lower in the peb4 mutant than in the wild-type strain. A Peb4 homolog has prolyl cis/trans-isomerase activity, suggesting that the loss of this activity in the mutant strain may be responsible for the repression of these proteins.     

Microbial film formation: dental plaque deposition on acrylic tiles using continuous culture techniques

A chemostat system has been developed to model the attachment of oral bacteria, and the subsequent development of plaque film, to acrylic surfaces immersed in steady state cultures. Plaque was removed from the teeth and gingival margin of volunteers who refrained from oral hygiene for at least 72 h. Samples were pooled and inoculated into a complex growth medium maintained at 37 degrees C. Glucose-limited continuous culture was established at a dilution rate of 0.05/h and at pH 7.0. Microbiological analysis of the culture indicated that a complex community of oral bacteria was established, typical of that found in dental plaque. Acrylic tiles were immersed in the fermenter through a modified fermenter head and incubated therein for up to 21 d. Scanning electron microscopy showed that either side of the tiles contained a rough and a smooth surface and these initially favoured the attachment of fusiform bacteria, particularly on the rough surface. Cocci attached to those surfaces which were not heavily colonized by the fusiforms and eventually grew into and on the colonial sheets of the fusiforms.     

Microbial film formation: dental plaque deposition on acrylic tiles using continuous culture techniques

A chemostat system has been developed to model the attachment of oral bacteria, and the subsequent development of plaque film, to acrylic surfaces immersed in steady state cultures. Plaque was removed from the teeth and gingival margin of volunteers who refrained from oral hygiene for at least 72 h. Samples were pooled and inoculated into a complex growth medium maintained at 37C. Glucose-limited continuous culture was established at a dilution rate of 005/h and at pH 70. Microbiological analysis of the culture indicated that a complex community of oral bacteria was established, typical of that found in dental plaque. Acrylic tiles were immersed in the fermenter through a modified fermenter head and incubated therein for up to 21 d. Scanning electron microscopy showed that either side of the tiles contained a rough and a smooth surface and these initially favoured the attachment of fusiform bacteria, particularly on the rough surface. Cocci attached to those surfaces which were not heavily colonized by the fusiforms and eventually grew into and on the colonial sheets of the fusiforms.     

Colonization of cattle intestines by Campylobacter jejuni and Campylobacter lanienae

The location and abundance of Campylobacter jejuni and Campylobacter lanienae in the intestines of beef cattle were investigated using real-time quantitative PCR in two studies. In an initial study, digesta and tissue samples were obtained along the digestive tract of two beef steers known to shed C. jejuni and C. lanienae (steers A and B). At the time of slaughter, steer B weighed 540 kg, compared to 600 kg for steer A, yet the intestine of steer B (40.5 m) was 36% longer than the intestine of steer A (26.1 m). In total, 323 digesta samples (20-cm intervals) and 998 tissue samples (3.3- to 6.7-cm intervals) were processed. Campylobacter DNA was detected in the digesta and in association with tissues throughout the small and large intestines of both animals. Although C. jejuni and C. lanienae DNA were detected in both animals, only steer A contained substantial quantities of C. jejuni DNA. In both digesta and tissues of steer A, C. jejuni was present in the duodenum and jejunum. Considerable quantities of C. jejuni DNA also were observed in the digesta obtained from the cecum and ascending colon, but minimal DNA was associated with tissues of these regions. In contrast, steer B contained substantial quantities of C. lanienae DNA, and DNA of this bacterium was limited to the large intestine (i.e., the cecum, proximal ascending colon, descending colon, and rectum); the majority of tissue-associated C. lanienae DNA was present in the cecum, descending colon, and rectum. In a second study, the location and abundance of C. jejuni and C. lanienae DNA were confirmed in the intestines of 20 arbitrarily selected beef cattle. DNA of C. jejuni and C. lanienae were detected in the digesta of 57% and 95% of the animals, respectively. C. jejuni associated with intestinal tissues was most abundant in the duodenum, ileum, and rectum. However, one animal contributed disproportionately to the abundance of C. jejuni DNA in the ileum and rectum. C. lanienae was most abundant in the large intestine, and the highest density of DNA of this bacterium was found in the cecum. Therefore, C. jejuni colonized the proximal small intestine of asymptomatic beef cattle, whereas C. lanienae primarily resided in the cecum, descending colon, and rectum. This information could be instrumental in developing efficacious strategies to manage the release of these bacteria from the gastrointestinal tracts of cattle.     

Development of an ex vivo organ culture model using human gastro-intestinal tissue and Campylobacter jejuni

Campylobacter jejuni is an important food-borne pathogen. However, relatively little is understood regarding its pathogenesis, and research is hampered by the lack of a suitable model. Recently, a number of groups have developed assays to study the pathogenic mechanisms of C. jejuni using cell culture models. Here, we report the development of an ex vivo organ culture model, allowing for the maintenance of intestinal mucosal tissue, to permit more complex host-bacterium interactions to be studied. Ex vivo organ culture highlights the propensity for C. jejuni to adhere to mucosal tissue via the flagellum, either as discrete colonies or as multicellular units.     

Colonization of Cattle Intestines by Campylobacter jejuni and Campylobacter lanienae

The location and abundance of Campylobacter jejuni and Campylobacter lanienae in the intestines of beef cattle were investigated using real-time quantitative PCR in two studies. In an initial study, digesta and tissue samples were obtained along the digestive tract of two beef steers known to shed C. jejuni and C. lanienae (steers A and B). At the time of slaughter, steer B weighed 540 kg, compared to 600 kg for steer A, yet the intestine of steer B (40.5 m) was 36% longer than the intestine of steer A (26.1 m). In total, 323 digesta samples (20-cm intervals) and 998 tissue samples (3.3- to 6.7-cm intervals) were processed. Campylobacter DNA was detected in the digesta and in association with tissues throughout the small and large intestines of both animals. Although C. jejuni and C. lanienae DNA were detected in both animals, only steer A contained substantial quantities of C. jejuni DNA. In both digesta and tissues of steer A, C. jejuni was present in the duodenum and jejunum. Considerable quantities of C. jejuni DNA also were observed in the digesta obtained from the cecum and ascending colon, but minimal DNA was associated with tissues of these regions. In contrast, steer B contained substantial quantities of C. lanienae DNA, and DNA of this bacterium was limited to the large intestine (i.e., the cecum, proximal ascending colon, descending colon, and rectum); the majority of tissue-associated C. lanienae DNA was present in the cecum, descending colon, and rectum. In a second study, the location and abundance of C. jejuni and C. lanienae DNA were confirmed in the intestines of 20 arbitrarily selected beef cattle. DNA of C. jejuni and C. lanienae were detected in the digesta of 57% and 95% of the animals, respectively. C. jejuni associated with intestinal tissues was most abundant in the duodenum, ileum, and rectum. However, one animal contributed disproportionately to the abundance of C. jejuni DNA in the ileum and rectum. C. lanienae was most abundant in the large intestine, and the highest density of DNA of this bacterium was found in the cecum. Therefore, C. jejuni colonized the proximal small intestine of asymptomatic beef cattle, whereas C. lanienae primarily resided in the cecum, descending colon, and rectum. This information could be instrumental in developing efficacious strategies to manage the release of these bacteria from the gastrointestinal tracts of cattle.     

Transcription of the Campylobacter jejuni cell division gene ftsA

Abstract Long-lived radioactive waste will be buried several hundred meters below ground in metal canisters surrounded by a buffer of compacted bentonite. Sulfate-reducing bacteria present in the bentonite may induce canister corrosion by production of hydrogen sulfide. Here we show that survival of sulfate-reducing bacteria in bentonite depends on the availability of water and that compacting a high quality bentonite to a water activity ( a _w) of 0.96 was lethal for the species investigated.     

Detection and quantification of Campylobacter jejuni and Campylobacter coli using real-time multiplex PCR

Aims: We describe a real‐time quantitative multiplex polymerase chain reaction (qmPCR) assay to identify and discriminate between isolates of Campylobacter jejuni and Campylobacter coli. Methods and Results: Two novel sets of primers and hydrolysis probes were designed to amplify the unique DNA sequences within the hipO, ccoN and cadF genes that are specific to Camp. jejuni and Camp. coli. Using the designed optimized qmPCR assay conditions, the amplification efficiency is in range from 108 to 116%. These qmPCR assays are highly specific for Camp. jejuni and Camp. coli, as seen through testing of 40 Campylobacter strains and 17 non‐Campylobacter strains. In chicken juice and tap water models spiked with known quantities of Camp. jejuni, qmPCR detected 10²–10³ CFU ml^?1 within 4 h. Conclusions: The qmPCR assays developed in this study provide reliable and simultaneous detection and quantification of Camp. jejuni and Camp. coli, with good amplification reaction parameters. Significance and Impact of the Study: Following further validation, the qmPCR assay reported here has the potential to be applied to various sample types as an alternative and rapid methodology.     

Bile-induced 'pili' in Campylobacter jejuni are bacteria-independent artifacts of the culture medium

In 1996, it was reported that the enteric pathogen Campylobacter jejuni produces pilus-like appendages in response to bile salts such as deoxycholate (DOC), and that the formation of these appendages requires the putative peptidase PspA. Pili were known to be important virulence determinants in other pathogenic bacteria but had never before been observed for C. jejuni. We report here that these appendages are not pili, but are instead a bacteria-independent morphological artifact of the growth medium. Furthermore, the pspA gene is not required for their formation. Broth cultures containing a threshold concentration of DOC inoculated with no bacteria produced identical abundant, fibrous, pilus-like structures as those cultures that had been inoculated with C. jejuni. These fibres were also found in growth media from DOC-containing pspA:CmR mutant cultures. These results are consistent with the absence of candidate pilin monomers in protein gel analyses as well as the dearth of pilin-like genes and pilus formation gene clusters in the C. jejuni genome.     

The effect of different media on the survival and induction of stress responses by Campylobacter jejuni

The survival kinetics of Campylobacter jejuni strain CI 120 to a challenge of pH 4.5 was studied in seven different media. A medium effect was observed, showing up to a 5-log difference in stress resistance of cells. Strain variation in survival of C. jejuni was observed in Brucella broth (BBL). The ability of C. jejuni CI 120 to respond to a stress after growth in seven different media was also examined. An Adaptive Tolerance Response (ATR) was induced in only three of the seven media tested. The degree of resistance induced by the ATR varied between the different media. The production, during growth, of an extracellular component that confers stress resistance against subsequent acid challenge was observed in only four of seven media tested. Due to the direct effect of medium on stress/survival of C. jejuni, the results suggest that studies using different media may not be comparable.     

Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals

By using 50 unabsorbed antisera, we were able to serotype 272 (65.7%) of 414 thermotolerant campylobacters from wild and domestic animals, on the basis of heat-stable antigens identified by means of passive hemagglutination. Forty-two serotypes were recognized. The pattern of serotypes detected in the various animal species was compared to human clinical isolates by using the Czekanowski index (proportional similarity index). The highest degree of similarity to the clinical isolates was observed for the poultry isolates, followed by strains from wild birds, flies, and pigs (in order of decreasing similarity). The serotypes recovered most frequently from poultry (LAU 1 and LAU 2) were also most prevalent in Norwegian patients. In contrast, serotype LAU 35/44, the predominant porcine serotype, was never recovered from human clinical specimens. Flies captured in chicken farms and in piggeries harbored serotypes which were also commonly seen in chickens and pigs, respectively. Nine of the strains included in this study could not be ascribed to any defined species. All of these were resistant to nalidixic acid and did not produce H2S.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

The response to oxidative stress of Fusobacterium nucleatum grown in continuous culture

Fusobacterium nucleatum ATCC 10953 was grown in continuous culture and the atmosphere changed stepwise from nitrogen (anaerobiosis) to a mixture of air: oxygen (40:60). No significant differences in biomass were observed and the baseline low level of superoxide dismutase increased only slightly; catalase and peroxidase activities were never detected but the level of NADH oxidase increased more than three-fold when oxygen was introduced into the system. In relation to acidic end-products, the relative proportion of acetate increased while that of butyrate decreased. Due mainly, it would seem, to NADH oxidase activity, the culture maintained a low redox potential (E(h)=-274 mV) even under an atmosphere of 40% oxygen in air and dissolved oxygen was not detected. This may, in part, explain the protective role of F. nucleatum for anaerobes in both biofilm and planktonic phases of aerated, mixed cultures of oral bacteria.     

Construction of a ferritin-deficient mutant of Campylobacter jejuni: contribution of ferritin to iron storage and protection against oxidative stress

The ferritin-encoding gene (cft) of Campylobacter jejuni was cloned and sequenced. The nucleotide sequence of cft had a 501 bp open reading frame for a protein with 167 amino acids and a predicted molecular mass of 19180 Da, and showed a high similarity to that of Helicobacter pylori and Escherichia coli ferritin genes. To determine the biological function of ferritin in C. jejuni, a ferritin-deficient mutant was constructed. The growth of ferritin-deficient strain SNA1 was clearly inhibited under iron deprivation. The ferritin-deficient mutant was more sensitive to killing by H₂O₂ and paraquat than the isogenic parent strain. These findings demonstrate that ferritin in C. jejuni makes a significant contribution to both iron storage and protection from intracellular iron overload, and resulting iron-mediated oxidative stress.     

Analysis of the Campylobacter jejuni Genome by SMRT DNA Sequencing Identifies Restriction-Modification Motifs

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C) content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in DNA restriction-modification. A total of five putative methylation motifs were identified as well as the C. jejuni enzymes that could be responsible for the modifications. Peptides corresponding to the deduced amino acid sequence of the C. jejuni enzymes were identified using proteomics. This work sets the stage for studies to dissect the precise functions of the C. jejuni putative restriction-modification enzymes. Taken together, the data generated in this study contributes to our knowledge of the genomic content, methylation profile, and encoding capacity of C. jejuni.