Mycobacterium avium subsp. paratuberculosis inhibits gamma interferon-induced signaling in bovine monocytes: insights into the cellular mechanisms of Johne's disease.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle and may have implications for human health. Establishment of chronic infection by M. avium subsp. paratuberculosis depends on its subversion of host immune responses. This includes blocking the ability of infected macrophages to be activated by gamma interferon (IFN-γ) for clearance of this intracellular pathogen. To define the mechanism by which M. avium subsp. paratuberculosis subverts this critical host cell function, patterns of signal transduction to IFN-γ stimulation of uninfected and M. avium subsp. paratuberculosis-infected bovine monocytes were determined through bovine-specific peptide arrays for kinome analysis. Pathway analysis of the kinome data indicated activation of the JAK-STAT pathway, a hallmark of IFN-γ signaling, in uninfected monocytes. In contrast, IFN-γ stimulation of M. avium subsp. paratuberculosis-infected monocytes failed to induce patterns of peptide phosphorylation consistent with JAK-STAT activation. The inability of IFN-γ to induce differential phosphorylation of peptides corresponding to early JAK-STAT intermediates in infected monocytes indicates that M. avium subsp. paratuberculosis blocks responsiveness at, or near, the IFN-γ receptor. Consistent with this hypothesis, increased expression of negative regulators of the IFN-γ receptors SOCS1 and SOCS3 as well as decreased expression of IFN-γ receptor chains 1 and 2 is observed in M. avium subsp. paratuberculosis-infected monocytes. These patterns of expression are functionally consistent with the kinome data and offer a mechanistic explanation for this critical M. avium subsp. paratuberculosis behavior. Understanding this mechanism may contribute to the rational design of more effective vaccines and/or therapeutics for Johne's disease.

Stability, toxicity, and biological activity of host defense peptide BMAP28 and its inversed and retro-inversed isomers.

Host defense peptides (HDPs) contribute to immune defense through direct antimicrobial activity as well as modulation of host immune responses. While the antimicrobial activity of HDPs has been successfully exploited as topical antibiotics, their use as systemic immunomodulatory antimicrobials has been limited by their toxicity and biological instability. Peptide modification strategies to address these characteristics, while maintaining biological activity, are likely essential to capture the full therapeutic potential of HDPs. Here we investigate the stability, toxicity, and biological activity of the L, inversed (D), and retro-inversed (RI) isomers of BMAP28. The D and RI isomers both form symmetrically related structures to L BMAP28 and resist proteolytic degradation. With respect to toxicity, the considerable hemolytic activity of L BMAP28 is approximately halved with the D isomer and eliminated with RI BMAP28. Furthermore, while D BMAP28 maintains the same cytotoxicity profile against epithelial cells and monocytes as the natural peptide, RI BMAP28 is markedly less toxic against these cell types. As prophylactic antimicrobials, all three isomers significantly reduced bacterial loads [99.99% bacterial clearance by each peptide at the highest dose (20 mg kg(-1) )], when administered 18 h prior to challenge in a mouse model of peritoneal infection. This protection appears to be mediated through neutrophil recruitment and activation of macrophages for bacterial clearance. Collectively, the increased stability and retained biological activity of D and RI BMAP28 make these isomers attractive as antimicrobial therapeutics. In particular, the protection conferred by RI BMAP28, combined with its reduced toxicities, make it a strong candidate for further consideration.

Clusters of circulating Neisseria gonorrhoeae strains and association with antimicrobial resistance in Shanghai.

OBJECTIVES: (i) To distinguish Neisseria gonorrhoeae isolates in Shanghai by porB typing; (ii) to ascertain the congruence of porB DNA sequence typing with cases linked epidemiologically; (iii) to determine the association of specific PorB mutations with antimicrobial resistance to penicillin or tetracycline. METHODS: porB DNA sequences of 174 N. gonorrhoeae isolates, collected from 143 male patients and 31 female sexual partners in Shanghai were determined. Phylogenetic analysis was used to determine sequence associations and concordance with epidemiologically linked cases. PorB protein sequences were compared with the wild-type sequence to identify mutations associated with antimicrobial resistance to penicillin and tetracycline. RESULTS: porB1a genotypes comprised 27.0% of the isolates and included 15 distinct DNA sequences, while 73.0% of the isolates carried porB1b genotypes with 63 distinct DNA sequences. porB DNA sequence typing was congruent with patient-reported sexual contacts. In addition, porB DNA sequence analysis revealed a number of strains with identical DNA sequences not identified through traditional epidemiological methods. The porB1b isolates had a significantly higher percentage of chromosomally mediated resistance to tetracycline and higher MIC50s to penicillin and ciprofloxacin. G120K/A121D mutations were observed in 71.1% of PIB isolates and were associated with resistance to penicillin and/or tetracycline. The majority of the PIA isolates (82.1%) also carried G120D/A121G double mutations. The index of discrimination for porB DNA sequence analysis was 95%. CONCLUSIONS: The porB1b genotype was found to be predominant in Shanghai. porB DNA sequence typing was sufficiently discriminatory for differentiating N. gonorrhoeae isolates and was congruent with epidemiological linkages. Novel porB sequences of N. gonorrhoeae and novel mutations of PorB proteins were identified.

Antimicrobial susceptibility and molecular determinants of quinolone resistance in Neisseria gonorrhoeae isolates from Shanghai.

OBJECTIVES: To determine the antimicrobial susceptibility of Neisseria gonorrhoeae from Shanghai and to type the quinolone resistance-determining regions (QRDRs) of ciprofloxacin-resistant isolates. METHODS: N. gonorrhoeae isolates (n = 159) were consecutively collected from male patients in Shanghai and examined for their antimicrobial susceptibilities to penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone. The mutation profiles of the QRDRs of gyrA and parC were determined for 103 isolates including one susceptible isolate and one isolate with intermediate levels of susceptibility to ciprofloxacin. RESULTS: High percentages of the 159 isolates were resistant to ciprofloxacin (98.7%), penicillin (93.1%) and tetracycline (56.5%). Penicillinase-producing N. gonorrhoeae (PPNG, 37.8%) or penicillinase-producing/tetracycline-resistant N. gonorrhoeae (PP/TRNG, 13.8%) accounted for 51.6% of the isolates. Chromosomal resistance to penicillin was observed in 41.5% of the isolates. Tetracycline resistance was noted in 56.5% of the isolates with 20.1% carrying plasmid-mediated resistance and 36.4% being chromosomally resistant. All isolates were susceptible to ceftriaxone and spectinomycin, although a trend to decreased susceptibility was noted. QRDR mutations were observed in the 101 ciprofloxacin-resistant isolates and the one ciprofloxacin-intermediate isolate, in contrast to the ciprofloxacin-susceptible isolate tested. Mutations in the QRDRs comprised four predominant (65.0% of the 103 isolates) patterns of a total of 19 patterns. Mutations in parC were significantly associated with higher MICs of ciprofloxacin. CONCLUSIONS: Spectinomycin and ceftriaxone are currently recommended for the treatment of gonorrhoea in Shanghai. Although the present study indicates that these antimicrobials should remain effective, the identification of isolates with decreased susceptibility underscores the importance of ongoing antimicrobial susceptibility surveillance to monitor and respond to the emergence of resistant isolates.