‘To be,or not to be’—The dilemma of ‘silent’ antimicrobial resistance genes in bacteria

Antimicrobial resistance is a serious threat to public health that dramatically undermines our ability to treat bacterial infections. Microorganisms exhibit resistance to different drug classes by acquiring resistance determinants through multiple mechanisms including horizontal gene transfer. The presence of drug resistance genotypes is mostly associated with corresponding phenotypic resistance against the particular antibiotic. However, bacterial communities harbouring silent antimicrobial resistance genes—genes whose presence is not associated with a corresponding resistant phenotype do exist. Under suitable conditions, the expression pattern of such genes often revert and regain resistance and could potentially lead to therapeutic failure. We often miss the presence of silent genes, since the current experimental paradigms are focused on resistant strains. Therefore, the knowledge on the prevalence, importance and mechanism of silent antibiotic resistance genes in bacterial pathogens are very limited. Silent genes, therefore, provide an additional level of complexity in the war against drug-resistant bacteria, reminding us that not only phenotypically resistant strains but also susceptible strains should be carefully investigated. In this review, we discuss the presence of silent antimicrobial resistance genes in bacteria, their relevance and their importance in public health.     

Cytotoxic cell granule-mediated apoptosis. Characterization of the macromolecular complex of granzyme B with serglycin

We have recently shown that the physiological mediator of granule-mediated apoptosis is a macromolecular complex of granzymes and perforin complexed with the chondroitin-sulfate proteoglycan, serglycin (Metkar, S. S., Wang, B., Aguilar-Santelises, M., Raja, S. M., Uhlin-Hansen, L., Podack, E., Trapani, J. A., and Froelich, C. J. (2002) Immunity 16, 417-428). We now report our biophysical studies establishing the nature of granzyme B-serglycin (GrB.SG) complex. Dynamic laser light scattering studies establish that SG has a hydrodynamic radius of approximately 140 +/- 23 nm, comparable to some viral particles. Agarose mobility shift gels and surface plasmon resonance (SPR), show that SG binds tightly to GrB and has the capacity to hold 30-60 GrB molecules. SPR studies also indicate equivalent binding affinities (K(d) approximately 0.8 microm), under acidic (granule pH) and neutral isotonic conditions (extra-cytoplasmic pH), for GrB.SG interaction. Finally, characterization of GrB.SG interactions within granules revealed complexes of two distinct molecular sizes, one held approximately 4-8 molecules of GrB, whereas the other contained as many as 32 molecules of GrB or other granule proteins. These studies provide a firm biophysical basis for our earlier reported observations that the proapoptotic granzyme is exocytosed predominantly as a macromolecular complex with SG.     

Growth of Escherichia coli in iron-enriched medium increases HPI catalase activity

Escherichia coli has two catalases, HPI and HPII. HPI is induced during logarithmic growth in response to low concentrations of hydrogen peroxide. This induction is OxyR-dependent. On the other hand, HPII is not peroxide-inducible but is induced in entry to the stationary phase. We demonstrate here that E. coli displayed higher HPI catalase activity when compared to the cultures that were grown in a normal medium, if grown in a medium supplemented with iron-citrate. Iron supplementation had no effect on HPII catalase. This increase of HPI activity was OxyR-independent and not observed in a Deltafur mutant. The physiological significance of the increase of HPI activity is unclear, but it appears that the katG gene that codes for HPI catalase is among the genes that are regulated by Fur.     

Influence of the MexAB-OprM Multidrug Efflux System on Quorum Sensing in Pseudomonas aeruginosa

Pseudomonas aeruginosa nalB mutants which hyperexpress the MexAB-OprM multidrug efflux system produce reduced levels of several extracellular virulence factors known to be regulated by quorum sensing. Such mutants also produce less acylated homoserine lactone autoinducer PAI-1, consistent with an observed reduction in lasI expression. These data suggest that PAI-1 is a substrate for MexAB-OprM, and its resulting exclusion from cells hyperexpressing MexAB-OprM limits PAI-1-dependent activation of lasI and the virulence genes.     

The use of ubiquitin lysine mutants to characterize E2–E3 linkage specificity: Mass spectrometry offers a cautionary “tail”

Oligomeric ubiquitin structures (i.e. ubiquitin “chains”) may be formed through any of seven different lysine residues in the polypeptide, or via the amine group of Met 1. Different types of ubiquitin chains can confer very different biological outcomes to a protein substrate, yet the structural characteristics of E2s and E3s that determine ubiquitin linkage specificity remain poorly understood. In vitro autoubiquitylation assays combined with ubiquitin protein variants bearing individually mutated lysine residues (“K‐to‐R” mutants) have thus been widely used to characterize E2–E3 linkage specificity. However, how this type of assay compares to direct identification of ubiquitin linkage types using mass spectrometry (MS) has not been rigorously tested. Here, we characterize the linkage specificity of 12 different E2–E3 combinations using both approaches. The simple MS‐based method described here is more robust, requires less material and is less prone to bias introduced by, e.g. the use of mutant proteins with unknown effects on E1, E2 or E3 recognition, antibodies with uncharacterized epitopes, the low dynamic range of X‐ray film, and additional sources of experimental error. Indeed, our results suggest that the K‐to‐R assay be approached with some caution.     

Effect of methanol intoxication on spcific immune functions of albino rats

Our previous studies revealed that methanol intoxication significantly altered the non-specific immune functions in albino rats. The present investigation focuses on the effect of methanol on certain specific immune functions of cell mediated immunity such as footpad thickness, leukocyte migration inhibition test (LMI) and antibody levels. In addition, serum interleukins (IL-2, IL-4, TNF-alpha and IFN-gamma), and splenic lymphocyte subsets were measured after an immune challenge. The specific immune function tests were carried out in three different groups of albino rats, which include control, 15 and 30 days methanol intoxication. Our study reports that animal body weight, organ weight ratio, lymphoid cell counts, footpad thickness, antibody titer, IL-2, TNF-alpha, IFN-gamma, Pan T cell, CD4, macrophages, MHC class II molecule expression, and B cell counts were significantly decreased compared to control animals nevertheless, LMI, IL-4, and DNA single strand breakage were increased significantly. Plasma corticosterone level was significantly increased in the 15 days group whereas the 30 days methanol intoxication group showed considerable decrease in corticosterone level compared with control animals. Therefore, our investigation concluded that repeated exposure of methanol profoundly suppressed the cell mediated and humoral immune functions in albino rats.     

Analysis of Pathogenic Diversity of the Rice Bacterial Blight Pathogen (Xanthomonas oryzae pv. oryzae) in the Andaman Islands and Identification of Effective Resistance Genes

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in the tropics for which genetic resistance in the host plants is the only effective solution. This study aimed at identification of resistance gene combinations effective against Xoo isolates and fingerprinting of the Xoo isolates of Andaman Islands (India). Here, we report the reaction of 21 rice BB differentials possessing Xa1 to Xa21 genes individually and in different combinations to various isolates of pathogen collected from Andaman Islands. Pathological screening results of 14 isolates revealed that among individual genes tested across 2 years, Xa4, Xa7 and Xa21 conferred resistance reaction across all isolates, whereas among combinations, IRBB 50 (Xa4 + xa5), IRBB 52 (Xa4 + Xa21) and IRBB 60 (Xa4 + xa5 + xa13 + Xa21) conveyed effective resistance against tested isolates. The nature of genetic diversity among four isolates selected on the basis of geographical isolation in the islands was studied through DNA finger printing. The RAPD primers S111, S119, S1117, S1109, S1103, S109 and S105 were found to be better indicators of molecular diversity among isolates than JEL primers. The diversity analysis grouped 14 isolates into three major clusters based on disease reaction wherein isolate no. 8 was found the most divergent as well as highly virulent. The remaining isolates were classified into two distinct groups. The importance of the study in the context of transfer of resistance gene(s) in the local cultivars specifically for tropical island conditions is presented and discussed.