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Biomedical subjects

R Srikumar

Publications and source records attributed to R Srikumar.

17 recordsLinked to original sources
PubMed2006

Effect of methanol intoxication on specific 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.

Animals↗
PubMed2001

Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa biofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance of P. aeruginosa biofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexA and mexC in the developing biofilm. Remarkably, expression of mexAB-oprM and mexCD-oprJ was not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprM and mexCD-oprJ expression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosa biofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of P. aeruginosa biofilms.

Anti-Bacterial Agents↗
PubMed2001

Multidrug efflux in Pseudomonas aeruginosa: components, mechanisms and clinical significance.

Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance during antibiotic therapy. Much of this resistance is promoted by highly homologous three-component efflux systems of broad substrate specificity, of which four have been identified to date. These include MexA-Mexs-OprM and MexX-MexY-OprM, which are expressed constitutively in wild type cells and, thus, provide for intrinsic multidrug resistance, and MexC-MexD-OprJ and MexE-MexF-OprN, whose expression so far has only been seen in acquired multidrug resistant mutant strains. Additional homologues of these efflux systems are identifiable in the recently released genome sequence, though their roles, if any, in antimicrobial efflux are unknown. These tripartite pumps are composed of an integral cytoplasmic membrane drug-proton antiporter of the resistance-nodulation-cell division (RND) family of exporters, a channel-forming outer membrane efflux protein (or outer membrane factor [OMF]) and a periplasmic membrane fusion protein (MFP) that links the other two. In addition to a number of antimicrobials of clinical significance, these pumps also export dyes, detergents, disinfectants, organic solvents and acylated homoserine lactones involved in quorum-sensing. While the natural functional of these pumps remains undefined, the fact that they contribute to antimicrobial resistance in P. aeruginosa makes them reasonable targets for therapeutic intervention.

Amino Acid Sequence↗
PubMed2000

Influence of mutations in the mexR repressor gene on expression of the MexA-MexB-oprM multidrug efflux system of Pseudomonas aeruginosa.

Several nalB-type multidrug-resistant mutants of Pseudomonas aeruginosa overexpressed MexAB-OprM and carried mutations in the local regulatory gene, mexR. Others, dubbed nalC types, carried mutations elsewhere and overexpressed MexAB-OprM less extensively than the nalB strains. Available evidence showed that MexR acted solely as repressor. Disruption of the mexR gene at various places suggested that the 5' end of mexR may be a part of the mexAB-oprM promoter.

Drug Resistance, Microbial↗
PubMed1999

Contribution of the MexAB-OprM multidrug efflux system to the beta-lactam resistance of penicillin-binding protein and beta-lactamase-derepressed mutants of Pseudomonas aeruginosa.

Deletion of the mexAB-oprM multidrug efflux operon substantially compromised the beta-lactam resistance of beta-lactamase-derepressed mutants of Pseudomonas aeruginosa, although it had only a modest impact on resistance of a penicillin-binding protein mutant. This highlights the multifactorial nature of beta-lactam resistance in this organism. Moreover, the contribution of efflux to the net resistance seen in some beta-lactam-resistant mutants suggests that inhibition of MexAB-OprM-mediated drug efflux might be an effective approach to overcoming beta-lactam resistance attributed to efflux as well as to other mechanisms of beta-lactam resistance.

Anti-Bacterial Agents↗
PubMed1998

Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD-OprJ in a multidrug-sensitive Escherichia coli strain.

The mexCD-oprJ and mexAB-oprM operons encode components of two distinct multidrug efflux pumps in Pseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosa efflux pump components in E. coli strains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the delta acrAB strain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosa efflux genes. E. coli KZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several beta-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to beta-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that beta-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coli KZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most beta-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains of P. aeruginosa. Surprisingly, E. coli KZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolC mutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their substrate specificity in E. coli.

Bacterial Outer Membrane Proteins↗
PubMed1998

Beta-lactamase inhibitors are substrates for the multidrug efflux pumps of Pseudomonas aeruginosa.

The MexAB-OprM multidrug efflux system exports a number of antimicrobial compounds, including beta-lactams. In an attempt to define more fully the range of antimicrobial compounds exported by this system, and, in particular, to determine whether beta-lactamase inhibitors were also accommodated by the MexAB-OprM pump, the influence of pump status (its presence or absence) on the intrinsic antibacterial activities of these compounds and on their abilities to enhance beta-lactam susceptibility in intact cells was assessed. MIC determinations clearly demonstrated that all three compounds tested, clavulanate, cloxacillin, and BRL42715, were accommodated by the pump. Moreover, by using beta-lactams which were readily hydrolyzed by the Pseudomonas aeruginosa class C chromosomal beta-lactamase, it was demonstrated that elimination of the mexAB-oprM-encoded efflux system greatly enhanced the abilities of cloxacillin and BRL42715 (but not clavulanate) to increase beta-lactam susceptibility. With beta-lactams which were poorly hydrolyzed, however, the inhibitors failed to enhance beta-lactam susceptibility in MexAB-OprM+ strains, although BRL42715 did enhance beta-lactam susceptibility in MexAB-OprM- strains, suggesting that even with poorly hydrolyzed beta-lactams this inhibitor was effective when it was not subjected to efflux. MexEF-OprN-overexpressing strains, but not MexCD-OprJ-overexpressing strains, also facilitated resistance to beta-lactamase inhibitors, indicating that these compounds are also substrates for the MexEF-OprN pump. These data indicate that an ability to inactivate MexAB-OprM (and like efflux systems in other bacteria) will markedly enhance the efficacies of beta-lactam-beta-lactamase inhibitor combinations in treating bacterial infections.

Anti-Bacterial Agents↗
PubMed1998

Contribution of outer membrane efflux protein OprM to antibiotic resistance in Pseudomonas aeruginosa independent of MexAB.

A Pseudomonas aeruginosa strain carrying an insertion of an omega Hg interposon in the mexB gene (mexB::omega Hg; strain K879) produced markedly reduced but still detectable levels of OprM, the product of the third gene of the mexAB-oprM multidrug efflux operon. By using a lacZ transcriptional fusion vector, promoter activity likely responsible for OprM expression in the mexB::omega Hg mutant was identified upstream of oprM. Introduction of the oprM gene, but not the mexAB genes, into a P. aeruginosa multidrug-susceptible delta mexAB-oprM mutant increased resistance to quinolones, cephalosporins, erythromycin, and tetracycline. A delta mexAB-oprM strain carrying the oprM gene accumulated markedly less antibiotic than the deletion strain without oprM. Antibiotic accumulation by the MexAB- OprM+ strain was markedly enhanced upon treatment of cells with the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that MexAB-independent OprM function likely involves an efflux process. Moreover, pretreatment of cells with CCCP prior to the accumulation assay abrogated any differences in accumulation levels between the MexAB- OprM+ and MexAB- OprM- strains, indicating that reduced drug accumulation by the OprM+ strain (in the absence of CCCP) cannot be due to OprM-mediated reduction in outer membrane permeability. It appears, therefore, the OprM can be expressed and function in a drug efflux capacity independent of MexAB.

Bacterial Outer Membrane Proteins↗
PubMed1998

Influence of the TonB energy-coupling protein on efflux-mediated multidrug resistance in Pseudomonas aeruginosa.

TonB couples the energized state of the cytoplasmic membrane to the operation of outer membrane receptors responsible for Fe(III) siderophore uptake across the outer membrane of gram-negative bacteria. A tonB mutant of Pseudomonas aeruginosa deficient in iron siderophore uptake was shown in the present study to be hypersusceptible to a wide variety of antibiotics, reminiscent of the phenotype of mutants defective in the mexAB-oprM antibiotic efflux operon. This was not related to influences of a tonB mutation on the iron status of the cell, and indeed, intrinsic antibiotic susceptibility and mexAB-oprM expression were unaffected by iron levels in the growth medium. The presence of tonB on a multicopy plasmid increased the level of resistance of a MexAB-OprM+ strain but not that of a MexAB-OprM- strain to a variety of antimicrobial agents. mexAB-oprM expression was not, however, altered in a tonB deletion mutant, indicating that any influence of TonB on MexAB-OprM-mediated multidrug resistance was at the level of pump activity. Consistent with this, drug accumulation assays revealed that the tonB deletion mutant exhibited decreased levels of drug efflux. Still, the multidrug resistance of a nalB strain was not wholly abrogated by a tonB mutation, indicating that it is likely not an essential component of the efflux apparatus. Similarly, elimination of tonB from an nfxB strain only partially compromised MexCD-OprJ-mediated multidrug resistance. Intriguingly, the drug susceptibility of a mexAB-oprM deletion strain was increased following deletion of tonB, suggesting that TonB may also influence antibiotic resistance mediated by determinants other than MexAB-OprM (and MexCD-OprJ). Thus, TonB plays an important role in both intrinsic and acquired antibiotic resistance in P. aeruginosa.

Anti-Bacterial Agents↗
PubMed1998

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.

4-Butyrolactone↗
PubMed1997

Porins of Haemophilus influenzae type b mutated in loop 3 and in loop 4.

Porin (341 amino acids; mass of 37,782 Da) in the outer membrane of Haemophilus influenzae type b (Hib) permits diffusion into the periplasm of small solutes up to a molecular mass of 1400 Da. Molecular modeling of Hib porin identified its structural similarities to OmpF of Escherichia coli and disclosed for Hib porin a shorter length of loop 3 and a longer length of loop 4. By site-directed mutagenesis of the porin gene ompP2, mutant porins were constructed to contain 6 or 12 amino acid deletions either in loop 3 or in surface-exposed loop 4. Wild type Hib porin and mutant porins were expressed in a nontypeable H. influenzae strain deleted for the ompP2 gene. The mutant porins were purified and reconstituted into planar bilayers, tested for channel formation and compared with wild type Hib porin. Mutant Haemophilus porin possessing a 6-amino acid deletion in loop 3 displayed a broad distribution of single channel conductance values, while deletion of 12 amino acids from the same loop destabilized the porin channel. By comparison, deletion of 6 or of 12 amino acids from loop 4 of Hib porin resulted in an increased single channel conductance (1.15 and 1.05 nanosiemens, respectively) compared with wild type Hib porin (0. 85 nanosiemens). The C3 epitope of the poliovirus VP1 capsid protein was inserted either into loop 3 or into loop 4 of Hib porin. By flow cytometry, the C3 epitope was detected as surface-exposed in strains expressing C3 insertion in loop 4; in strains expressing C3 insertion in loop 3, the epitope was inaccessible. We propose that loop 4 of Hib porin, although surface-accessible, is oriented toward the central axis of the pore and that deletions in this loop increase the single channel conductance by widening the pore entrance.

Amino Acid Sequence↗
PubMed1997

Inner membrane efflux components are responsible for beta-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa.

A major feature of the MexAB-OprM multidrug efflux pump which distinguishes it from the MexCD-OprJ and MexEF-OprN multidrug efflux systems in Pseudomonas aeruginosa is its ability to export a wide variety of beta-lactam antibiotics. Given the periplasmic location of their targets it is feasible that beta-lactams exit the cell via the outer membrane OprM without interaction with MexA and MexB, though the latter appear to be necessary for OprM function. To test this, chimeric MexAB-OprJ and MexCD-OprM efflux pumps were reconstituted in delta mexCD delta oprM and delta mexAB delta oprJ strains, respectively, and the influence of the exchange of outer membrane components on substrate (i.e., beta-lactam) specificity was assessed. Both chimeric pumps were active in antibiotic efflux, as evidenced by their contributions to resistance to a variety of antimicrobial agents, although there was no change in resistance profiles relative to the native pumps, indicating that OprM is not the determining factor for the beta-lactam specificity of MexAB-OprM. Thus, one or both of inner membrane-associated proteins MexA and MexB are responsible for drug recognition, including recognition of beta-lactams.

Bacterial Outer Membrane Proteins↗
PubMed1996

Purification and refolding of recombinant Haemophilus influenzae type b porin produced in Bacillus subtilis.

The major diffusion channel in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; Mr 37 782). The Hib porin gene was cloned and overexpressed in Bacillus subtilis. Recombinant Hib porin (Bac porin), having aggregated into inclusion bodies, was purified under denaturing conditions and subsequently refolded. To compare Bac porin that is intrinsically devoid of lipooligosaccharides versus native Hib porin, the properties of Bac porin were assessed by the following four criteria: circular dichroism spectroscopy, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by an anti-Hib porin monoclonal antibody. We conclude that in the absence of lipooligosaccharides, Bac porin was refolded into a functional form which closely resembled the structure of Hib porin.

Animals↗
PubMed1993

Immunological properties of recombinant porin of Haemophilus influenzae type b expressed in Bacillus subtilis.

The major surface-located, channel-forming protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; M(r), 37,782). In order to generate Hib porin that is devoid of lipooligosaccharides and capsular polysaccharide, the Hib porin gene ompP2 was subcloned into a plasmid vector and recombinant Hib porin was expressed in Bacillus subtilis. Recombinant porin was produced in large quantities in B. subtilis and formed intracellular inclusion bodies. Recombinant porin was extracted from inclusion bodies and shown to be active in forming pores in synthetic black lipid membranes. However, these pores demonstrated different pore characteristics than wild-type Hib porin. Mouse hyperimmune sera against recombinant porin were generated and subjected to epitope scanning with a library of 336 overlapping synthetic hexapeptides that corresponded to the entire sequence of Hib porin. The epitope specificities of the anti-recombinant porin antibodies were similar to those of antibodies against Hib porin: selected regions near the amino terminus which include a buried loop in the native structure of Hib porin were more immunogenic than regions at the carboxy terminus. Although some mouse anti-recombinant porin antibodies mediated complement-dependent binding to Hib by polymorphonuclear leucocytes in opsonophagocytosis assays, the antibodies were not bactericidal, nor did they abrogate bacteremia in the infant rat model of infection. It was concluded that the native state of Hib porin is required for the generation of a protective immune response against the bacterium.

Animals↗
PubMed1992

Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities.

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38,000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial cell surface, seven mouse monoclonal anti-Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104-139; domain (ii) amino acids 162-174; and domain (iii), amino acids 267-341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement-mediated lysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface-binding, they were neither bactericidal nor protective.

Amino Acid Sequence↗
PubMed1992

Antigenic sites on porin of Haemophilus influenzae type b: mapping with synthetic peptides and evaluation of structure predictions.

The major surface-located protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin, molecular mass, 38 kDa, 341 amino acids. To define precisely the molecular reactivities of nine mouse monoclonal antibodies (MAbs) against Hib porin, overlapping hexapeptides corresponding to the entire sequence of porin were synthesized. The epitopes recognized by the MAbs were mapped by enzyme-linked immunosorbent assay to stretches of 6 to 11 amino acids. Antigenic sites between amino acids 112 and 126, 148 and 153, 162 and 172, and 318 and 325 were identified. The antigenic sites between amino acids 162 and 172 and between amino acids 318 and 325 were determined by flow cytometry to be on the bacterial cell surface. Four MAbs, POR.2, POR.3, POR.4, and POR.5, that react with amino acids 162 to 172 were able to discriminate among porins from the three major outer membrane protein subtypes of Hib, i.e., 1H, 2L, and 6U. A model for the topological organization of Hib porin was created by calculating the hydrophobicity, amphiphilicity, and turn propensity in its amino acid sequence. Determination of the molecular reactivities of the anti-Hib porin MAbs provided substantive evidence for the orientation of selected regions of porin in the outer membrane of Hib.

Amino Acid Sequence↗