Chloe E. James

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria.

Gram-negative bacteria are responsible for a large proportion of antibiotic-resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channels.

HIV protease inhibitors are substrates for OATP1A2, OATP1B1 and OATP1B3 and lopinavir plasma concentrations are influenced by SLCO1B1 polymorphisms

Objective OATP1B1 and OATP1B3 are major hepatic drug transporters whilst OATP1A2 is mainly located in the brain but is also located in liver and several other organs. These transporters affect the distribution and clearance of many endobiotics and xenobiotics and have been reported to have functional single nucleotide polymorphisms (SNPs). We have assessed the substrate specificities of these transporters for a panel of antiretrovirals and investigated the effects of SNPs within these transporters on the pharmacokinetics of lopinavir. Methods SLCO1A2, SLCO1B1 and SLCO1B3 were cloned, verified and used to generate cRNA for use in the Xenopuslaevis oocyte transport system. Using the oocyte system, antiretrovirals were tested for their substrate specificities. Plasma samples (n=349) from the Liverpool therapeutic drug monitoring registry were genotyped for SNPs in SLCO1A2, SLCO1B1 and SLCO1B3 and associations between SNPs and lopinavir plasma concentrations were analysed. Result Antiretroviral protease inhibitors, but not non-nucleoside reverse transcriptase inhibitors, are substrates for OATP1A2, OATP1B1 and OATP1B3. Furthermore, ritonavir was not an inhibitor of OATP1B1. The 521T>C polymorphism in SLCO1B1 was significantly associated with higher lopinavir plasma concentrations. No associations were observed with functional variants of SLCO1A2 and SLCO1B3. Conclusion These data add to our understanding of the factors that contribute to variability in plasma concentrations of protease inhibitors. Further studies are now required to confirm the association of SLCO1B1 521T>C with lopinavir plasma concentrations and to assess the influence of other polymorphisms in the SLCO family.

Membrane Permeability and Regulation of Drug “Influx and Efflux” in Enterobacterial Pathogens

In Enterobacteriaceae, membrane permeability is a key in the level of susceptibility to antibiotics. Modification of the bacterial envelope by decreasing the porin production or increasing the expression of efflux pump systems has been reported. These phenomena are frequently associated with other resistance mechanisms such as alteration of antibiotics or modification of the drug targets, in various clinical isolates showing a Multi Drug Resistant phenotype (MDR). In Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Salmonella enterica several genes and external factors are involved in the emergence of MDR isolates. These bacterial isolates exhibit a noticeable reduction of functional porins per cell due to a decrease, a complete shutdown of synthesis, or the expression of an altered porin and a high expression of efflux systems (e.g. overexpression of the pump). The combined action of these mechanisms during an infection confers a significant decrease in bacterial sensitivity to antibiotherapy ensuring dissemination and colonization of the patient and favours the acquisition of additional mechanisms of resistance. MarA and ramA are involved in a complex regulation cascade controlling membrane permeability and actively participate in the triggering of the MDR phenotype. Mutations in regulator genes have been shown to induce the overproduction of efflux and the down-regulation of porin synthesis. In addition, various compounds such as salicylate, imipenem or chloramphenicol are able to activate the MDR response. This phenomenon has been observed both in vitro during culture of bacteria in the presence of drugs and in vivo during antibiotic treatment of infected patients. These effectors activate the expression of specific global regulators, marA, ramA, or target other genes located downstream in the regulation cascade.

Intrinsic apoptotic pathways of gingival epithelial cells modulated by Porphyromonas gingivalis

Porphyromonas gingivalis can inhibit chemically induced apoptosis in primary cultures of gingival epithelial cells through blocking activation of the effector caspase‐3. The anti‐apoptotic phenotype of P. gingivalis is conserved across strains and does not depend on the presence of fimbriae, as fimbriae‐deficient mutants and a naturally occurring non‐fimbriated strain were able to impede apoptosis. To dissect the survival pathways modulated by P. gingivalis, protein and gene expression of a number of components of apoptotic death pathways were investigated. P. gingivalis infection of epithelial cells resulted in the phosphorylation of JAK1 and Stat3. Quantitative real‐time reverse transcription polymerase chain reaction showed that expression of Survivin and Stat3 itself, targets of activated Stat3, were elevated in P. gingivalis‐infected cells. siRNA knockdown of JAK1, in combination with knockdown of Akt, abrogated the ability of P. gingivalis to block apoptosis. In contrast, cIAP‐1 and cIAP‐2 were not differentially regulated at either the protein or mRNA levels by P. gingivalis. One mechanism by which P. gingivalis can block apoptotic pathways in gingival epithelial cells therefore is through manipulation of the JAK/Stat pathway that controls the intrinsic mitochondrial cell death pathways. Induction of a pro‐survival phenotype may prevent programmed host cell death and aid survival of P. gingivalis within gingival epithelial cells.

Streptococcus gordonii utilizes several distinct gene functions to recruit Porphyromonas gingivalis into a mixed community

Dental plaque biofilm formation proceeds through a developmental pathway initiated by the attachment of pioneer organisms, such as Streptococcus gordonii, to tooth surfaces. Through a variety of synergistic interactions, pioneer organisms facilitate the colonization of later arrivals including Porphyromonas gingivalis, a potential periodontal pathogen. We have investigated genes of S. gordonii required to support a heterotypic biofilm community with P. gingivalis. By screening a plasmid integration library of S. gordonii, genes were identified that are crucial for the accumulation of planktonic P. gingivalis cells into a multispecies biofilm. These genes were further investigated by specific mutation and complementation analyses. The biofilm‐associated genes can be grouped into broad categories based on putative function as follows: (i) intercellular or intracellular signalling (cbe and spxB), (ii) cell wall integrity and maintenance of adhesive proteins (murE, msrA and atf), (iii) extracellular capsule biosynthesis (pgsA and atf), and (iv) physiology (gdhA, ccmA and ntpB). In addition, a gene for a hypothetical protein was identified. Biofilm visualization and quantification by confocal microscopy confirmed the role of these genes in the maturation of the multispecies community, including biofilm architectural development. The results suggest that S. gordonii governs the development of heterotypic oral biofilms through multiple genetic pathways.

Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage.

ABSTRACT A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number ofShigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.

LuxS Involvement in the Regulation of Genes Coding for Hemin and Iron Acquisition Systems in Porphyromonas gingivalis

ABSTRACT The periodontal pathogen Porphyromonas gingivalis employs a variety of mechanisms for the uptake of hemin and inorganic iron. Previous work demonstrated that hemin uptake in P. gingivalis may be controlled by LuxS-mediated signaling. In the present study, the expression of genes involved in hemin and iron uptake was determined in parent and luxS mutant strains by quantitative real-time reverse transcription-PCR. Compared to the parental strain, the luxS mutant showed reduced levels of transcription of genes coding for the TonB-linked hemin binding protein Tlr and the lysine-specific protease Kgp, which can degrade host heme-containing proteins. In contrast, there was up-regulation of the genes for another TonB-linked hemin binding protein, HmuR; a hemin binding lipoprotein, FetB; a Fe2+ ion transport protein, FeoB1; and the iron storage protein ferritin. Differential expression of these genes in the luxS mutant was maximal in early-exponential phase, which corresponded with peak expression of luxS and AI-2 signal activity. Complementation of the luxS mutation with wild-type luxS in trans rescued expression of hmuR. Mutation of the GppX two-component signal transduction pathway caused an increase in expression of luxS along with tlr and lower levels of message for hmuR. Moreover, expression of hmuR was repressed, and expression of tlr stimulated, when the luxS mutant was incubated with AI-2 partially purified from the culture supernatant of wild-type cells. A phenotypic outcome of the altered expression of genes involved in hemin uptake was impairment of growth of the luxS mutant in hemin-depleted medium. The results demonstrate a role of LuxS/AI-2 in the regulation of hemin and iron acquisition pathways in P. gingivalis and reveal a novel control pathway for luxS expression.

Immunity profiles of wild-type and recombinant shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens.

ABSTRACT The pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype O157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx1 and/or stx2) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx1 or stx2 genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage λ, and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.

How β-Lactam Antibiotics Enter Bacteria: A Dialogue with the Porins

Background Multi-drug resistant (MDR) infections have become a major concern in hospitals worldwide. This study investigates membrane translocation, which is the first step required for drug action on internal bacterial targets. β-lactams, a major antibiotic class, use porins to pass through the outer membrane barrier of Gram-negative bacteria. Clinical reports have linked the MDR phenotype to altered membrane permeability including porin modification and efflux pump expression. Methodology/Principal Findings Here influx of β-lactams through the major Enterobacter aerogenes porin Omp36 is characterized. Conductance measurements through a single Omp36 trimer reconstituted into a planar lipid bilayer allowed us to count the passage of single β-lactam molecules. Statistical analysis of each transport event yielded the kinetic parameters of antibiotic travel through Omp36 and distinguishable translocation properties of β-lactams were quantified for ertapenem and cefepime. Expression of Omp36 in an otherwise porin-null bacterial strain is shown to confer increases in the killing rate of these antibiotics and in the corresponding bacterial susceptibility. Conclusions/Significance We propose the idea of a molecular “passport” that allows rapid transport of substrates through porins. Deciphering antibiotic translocation provides new insights for the design of novel drugs that may be highly effective at passing through the porin constriction zone. Such data may hold the key for the next generation of antibiotics capable of rapid intracellular accumulation to circumvent the further development MDR infections.

Role of environmental survival in transmission of Campylobacter jejuni

Campylobacter species are the most common cause of bacterial gastroenteritis, with C. jejuni responsible for the majority of these cases. Although it is clear that livestock, and particularly poultry, are the most common source, it is likely that the natural environment (soil and water) plays a key role in transmission, either directly to humans or indirectly via farm animals. It has been shown using multilocus sequence typing that some clonal complexes (such as ST-45) are more frequently isolated from environmental sources such as water, suggesting that strains vary in their ability to survive in the environment. Although C. jejuni are fastidious microaerophiles generally unable to grow in atmospheric levels of oxygen, C. jejuni can adapt to survival in the environment, exhibiting aerotolerance and starvation survival. Biofilm formation, the viable but nonculturable state, and interactions with other microorganisms can all contribute to survival outside the host. By exploiting high-throughput technologies such as genome sequencing and RNA Seq, we are well placed to decipher the mechanisms underlying the variations in survival between strains in environments such as soil and water and to better understand the role of environmental persistence in the transmission of C. jejuni directly or indirectly to humans.