Publications

Thermophilic Campylobacter, particularly Campylobacter jejuni, is one of the major foodborne human pathogens of animal origin. Reliable and sensitive typing tools are required for understanding the epidemiology and ecology of this zoonotic bacteria agent. Currently, several molecular typing methods are available for differentiating Campylobacter strains, but each of them has limitations. Our previous study revealed that considerable sequence polymorphism exists in the cmp gene encoding the major outer membrane protein of Campylobacter and suggested that sequence variation of cmp may be utilized for discrimination of Campylobacter strains. In this study, we evaluated the feasibility of the cmp-based typing tool, using pulsed-field gel electrophoresis (PFGE) as the "gold" standard for comparison. The cmp alleles were sequenced from multiple Campylobacter strains, grouped, and compared with the PFGE profiles of these strains using Bionumerics. Results showed that 43 cmp sequence types and 43 PFGE types existed among the 60 Campylobacter isolates. Typeability of these strains is 100% using either the cmp-based method or PFGE. The discrimination indices are 0.973 for the cmp-based method and 0.969 for PFGE, respectively. The cmp sequence types are 77.6% congruent with the PFGE types. These results indicate that the cmp-based typing is a simple, yet highly discriminatory approach for molecular differentiation of C. jejuni strains.

The novel class Ib molecule MR1 is highly conserved in mammals, particularly in its alpha1/alpha2 domains. Recent studies demonstrated that MR1 expression is required for development and expansion of a small population of T cells expressing an invariant T cell receptor (TCR) alpha chain called mucosal-associated invariant T (MAIT) cells. Despite these intriguing properties it has been difficult to determine whether MR1 expression and MAIT cell recognition is ligand-dependent. To address these outstanding questions, monoclonal antibodies were produced in MR1 knock-out mice immunized with recombinant MR1 protein, and a series of MR1 mutations were generated at sites previously shown to disrupt the ability of class Ia molecules to bind peptide or TCR. Here we show that 1) MR1 molecules are detected by monoclonal antibodies in either an open or folded conformation that correlates precisely with peptide-induced conformational changes in class Ia molecules, 2) only the folded MR1 conformer activated 2/2 MAIT hybridoma cells tested, 3) the pattern of MAIT cell activation by the MR1 mutants implies the MR1/TCR orientation is strikingly similar to published major histocompatibility complex/alphabetaTCR engagements, 4) all the MR1 mutations tested and found to severely reduce surface expression of folded molecules were located in the putative ligand binding groove, and 5) certain groove mutants of MR1 that are highly expressed on the cell surface disrupt MAIT cell activation. These combined data strongly support the conclusion that MR1 has an antigen presentation function.

Campylobacter jejuni, a major foodborne human pathogen, has become increasingly resistant to fluoroquinolone (FQ) antimicrobials. By using clonally related isolates and genetically defined mutants, we determined the fitness of FQ-resistant Campylobacter in chicken (a natural host and a major reservoir for C. jejuni) in the absence of antibiotic selection pressure. When monoinoculated into the host, FQ-resistant and FQ-susceptible Campylobacter displayed similar levels of colonization and persistence in the absence of FQ antimicrobials. The prolonged colonization in chickens did not result in loss of the FQ resistance and the resistance-conferring point mutation (C257 –> T) in the gyrA gene. Strikingly, when coinoculated into chickens, the FQ-resistant Campylobacter isolates outcompeted the majority of the FQ-susceptible strains, indicating that the resistant Campylobacter was biologically fit in the chicken host. The fitness advantage was not due to compensatory mutations in the genes targeted by FQ and was linked directly to the single point mutation in gyrA, which confers on Campylobacter a high-level resistance to FQ antimicrobials. In certain genetic backgrounds, the same point mutation entailed a biological cost on Campylobacter, as evidenced by its inability to compete with the FQ-susceptible Campylobacter. These findings provide a previously undescribed demonstration of the profound effect of a resistance-conferring point mutation in gyrA on the fitness of a major foodborne pathogen in its natural host and suggest that the rapid emergence of FQ-resistant Campylobacter on a worldwide scale may be attributable partly to the enhanced fitness of the FQ-resistant isolates.

Using laboratory challenge experiments, we examined whether Campylobacter-specific maternal antibody (MAB) plays a protective role in young chickens, which are usually free of Campylobacter under natural production conditions. Kinetics of C. jejuni colonization were compared by infecting 3-day-old broiler chicks, which were naturally positive for Campylobacter-specific MAB, and 21-day-old broilers, which were negative for Campylobacter-specific MAB. The onset of colonization occurred much sooner in birds challenged at the age of 21 days than it did in the birds inoculated at 3 days of age, which suggested a possible involvement of specific MAB in the delay of colonization. To further examine this possibility, specific-pathogen-free layer chickens were raised under laboratory conditions with or without Campylobacter infection, and their 3-day-old progenies with (MAB(+)) or without (MAB(-)) Campylobacter-specific MAB were orally challenged with C. jejuni. Significant decreases in the percentage of colonized chickens were observed in the MAB(+) group during the first week compared with the MAB(-) group. These results indicate that Campylobacter-specific MAB plays a partial role in protecting young chickens against colonization by C. jejuni. Presence of MAB in young chickens did not seem to affect the development of systemic immune response following infection with C. jejuni. However, active immune responses to Campylobacter occurred earlier and more strongly in birds infected at 21 days of age than those infected at 3 days of age. Clearance of Campylobacter infection was also observed in chickens infected at 21 days of age. Taken together, these findings (i) indicate that anti-Campylobacter MAB contributes to the lack of Campylobacter infection in young broiler chickens in natural environments and (ii) provide further evidence supporting the feasibility of development of immunization-based approaches for control of Campylobacter infection in poultry.

Outer membrane proteins (OMPs) of Gram-negative bacteria have diverse functions and are directly involved in the interaction with various environments encountered by pathogenic organisms. Thus, OMPs represent important virulence factors and play essential roles in bacterial adaptation to host niches, which are usually hostile to invading pathogens. Understanding the structure and functions of bacterial OMPs will facilitate the design of antimicrobial drugs and vaccines. In this paper, we will present a brief review on OMPs that contribute to bacterial adaptive responses including iron uptake, antimicrobial peptide resistance, serum resistance, and drug/bile resistance.