SummaryThe MtrC-MtrD-MtrE efflux pump system confers resistance to macrolide antibiotics and antimicrobial substances of the host innate defense. Clinical isolates with increased resistance to erythromycin and azithromycin frequently harbor mutations in the mtrR structural gene, which encodes a repressor of the mtrCDE operon, or the mtrR promoter region. The MtrC-MtrD-MtrE system is important for gonococcal survival in the murine genital tract, and derepression of the mtrCDE operon via deletion of mtrR confers increased fitness in vivo. Here we compared isogenic strains with naturally occurring mtrR locus mutations for differences in mtrCDE expression and pump-related phenotypes. Mutations upstream of mtrC, including those within the MtrR binding region and a novel mutation that increases mtrC RNA stability conferred the highest levels of derepression as measured by mtrCDE transcription and resistance to antibiotics, progesterone, and antimicrobial peptides. In contrast, mutations within the mtrR coding sequence conferred low to intermediate levels of derepression. In vivo, the mtr mutants were more fit than the wild type strain, the degree to which paralleled in vitro resistance gradients. These studies establish a hierarchy of mtrR locus mutations with regard to regulation of pump efflux, and suggest selection for more derepressed mutants may occur during mixed infections.

The Neisseria gonorrhoeae MtrC-MtrD-MtrE multidrug-resistance efflux pump expels macrolide antibiotics, penicillin, and antimicrobial effectors of the innate defense. Mutation of the mtrR locus, which encodes a transcriptional repressor of the mtrCDE operon, increases gonococcal resistance to these agents. Here we report that, in a mouse infection model, an mtrR mutant is more fit than the wild-type bacteria. Consistent with derepression of the mtrCDE operon as the primary reason for the fitness benefit, an mtrR,mtrE double mutant and an mtrE mutant showed no difference in survival phenotype. Gonococcal mutants deficient in MtrA, an activator of the mtrCDE operon, exhibited significantly reduced fitness in vivo, and mtrA mutants with spontaneous compensatory mtrR mutations were selected during infection. These results confirm the importance of the MtrC-MtrD-MtrE efflux-pump system during experimental gonococcal genital-tract infection and also illustrate an antibiotic-resistance mechanism that is accompanied by a fitness benefit rather than a fitness cost.

Cationic antimicrobial peptides (CAMPs), a component of the mammalian immune system, protect the host from bacterial infections. The roles of the Escherichia coli transcriptional regulators MarA, SoxS and Rob in susceptibility to these peptides were examined. Overexpression of marA, either in an antibiotic-resistant marR mutant or from a plasmid, decreased bacterial susceptibility to CAMPs. Overexpression of the soxS gene from a plasmid, which decreased susceptibility to antibiotics, unexpectedly caused no decrease in CAMP susceptibility; instead it produced increased susceptibility to different CAMPs. Deletion or overexpression of rob had little effect on CAMP susceptibility. The marRAB operon was upregulated when E. coli was incubated in sublethal amounts of CAMPs polymyxin B, LL-37 or human b-defensin-1; however, this upregulation required Rob. Deletion of acrAB increased bacterial susceptibility to polymyxin B, LL-37 and human b-defensin-1 peptides. Deletion of tolC yielded an even greater increase in susceptibility to these peptides and also led to increased susceptibility to human a-defensin-2. Inhibition of cellular proton-motive force increased peptide susceptibility for wild-type and acrAB deletion strains; however, it decreased susceptibility of tolC mutants. These findings demonstrate that CAMPs are both inducers of marA-mediated drug resistance through interaction with Rob and also substrates for efflux in E. coli. The three related transcriptional regulators show different effects on bacterial cell susceptibility to CAMPs.

A carbapenem-resistant clinical isolate of Escherichia coli, which lacked OmpF and OmpC porins, carried a marR mutation and expressed a functional yedS, a normally nontranslated gene. MarR and YedS are described here as having effects on the ability of this strain to resist carbapenems. Additionally, expression of YedS was regulated by the small RNA MicF in a MarA-dependent way. These findings illustrate how broadly bacteria can mutate within a selective clinical setting, in this case, resistance to carbapenems, by altering three porin genes and one regulatory gene. Carbapenems are broad-spectrum ␤-lactam antibiotics used for the treatment of multidrug-resistant Gram-negative pathogens (1-3). Carbapenem resistance most commonly arises through the acquisition of genes encoding carbapenemases, which hydrolyze carbapenems (3-5). The other chief mechanism of carbapenem resistance in Escherichia coli and other Enterobacteriaceae is decreased bacterial cell permeability due to loss or alteration of the outer membrane porins F and/or C (1, 6-8).The marRAB operon of E. coli encodes the MarR repressor, the transcriptional regulator MarA, and a putative small protein, MarB (9). MarR represses transcription of marRAB by binding to marO and negatively controlling MarA-dependent expression of other genes in the regulon (10, 11). Upon induction by a variety of compounds (12) or by mutation of marR or marO, the repressor is rendered inactive (10). The resulting overexpression of MarA produces antibiotic resistance by increasing the expression of the major multidrug efflux pump AcrAB-TolC (13, 14) and downregulating the outer membrane protein OmpF via the small RNA (sRNA) MicF (15,16). In this study, a carbapenem-resistant, noncarbapenemase-producing clinical isolate of E. coli from China (CH4) was investigated to determine the genetic basis for the carbapenem resistance phenotype.PCR amplification and sequencing using the primers marRfor (5=-ATTAGCGGCCGCATCGGTCAATTCAT) and marRrev (5=-ATAGGATCCTTACGGCTGCGGATGTA) revealed numerous mutations in the marR open reading frame (ORF) of strain CH4 and other clinical isolates from China (Table 1). We cloned ORFs containing the various marR mutations, using the primers marR-clone-For and marR-clone-Rev (17), into expression vector pET-13a (18), for which expression was controlled by the T7 promoter. Expression of T7 polymerase was induced from plasmid pACT7-Spc (19) via isopropyl-␤-D-thiogalactopyranoside (IPTG) in the reporter strain SPC-106, a marO-lacZ fusion that contains a ⌬marR mutation (12). Analysis of LacZ activity (11,20) showed that the Gly42Arg mutation in the CH4 marR gene did not complement the ⌬marR mutation in this reporter strain (Fig. 1), indicating that this mutation affected the activity of MarR.We then complemented the marR mutation in CH4 by transforming the strain with pET-marR wildtype and pACT7-Spc or pAC-MarRwt (17). MICs were determined. The data showed that the two expression vectors produced similar decreases in resistance in strain CH4 that were not seen wi...

Typing of the porB variable region (VR) is an epidemiological tool that classifies gonococcal strains based on sequence differences in regions of the porB gene that encode surface-exposed loops. The frequent isolation of certain porB VR types suggests that some porin sequences confer a selective advantage during infection and/or transmission. Alternatively, certain porin types may be markers of strains that are successful due to factors unrelated to porin. In support of the first hypothesis, here we show urogenital tract isolates representing the most common PIA VR types identified in an urban clinic in Baltimore, MD, over a 10-year period belonged to several different clonal types, as determined by pulsed-field gel electrophoresis (PFGE). Serum resistance, which was confirmed by factor H and C4b-binding protein binding studies, was more often associated with gonococcal the most common VR types. In contrast, three porin-independent phenotypes, namely, lactoferrin utilization, ␤-lactamase production, and multiple transferable resistance (Mtr), were segregated with the PFGE cluster and not with the VR type. Data combined with another PIA strain collection showed a strong correlation between serum resistance and the most common VR types. A comparison of VR typing hybridization patterns and nucleotide sequences of 12 porB1a genes suggests that certain porin loop 1, 3, 6, and/or 7 sequences may play a role in the serum resistance phenotype. We conclude that some PorB PIA sequences confer a survival or transmission advantage in the urogenital tract, perhaps via increased resistance to complement-mediated killing. The capacity of some porin types to evade a porin-specific adaptive immune response must also be considered.

Paralogous transcriptional regulators MarA, Rob, and SoxS act individually and together to control expression of more than 80 Escherichia coli genes. Deletion of marA, rob, and soxS from an E. coli clinical isolate prevents persistence beyond 2 days postinfection in a mouse model of pyelonephritis. We used microarray analysis to identify 242 genes differentially expressed between the triple deletion mutant and its parent strain at 2 days postinfection in the kidney. One of these, znuC of the zinc transport system ZnuACB, displayed decreased expression in the triple mutant compared to that in the parental strain, and deletion of znuC from the parental strain reduced persistence. The marA rob soxS triple deletion mutant was less viable in vitro under limited-Zn and Zn-depleted conditions, while disruption of znuC caused a reduction in the growth rates for the parental and triple mutant strains to equally low levels under limited-Zn or Zn-depleted conditions. Complementation of the triple mutant with soxS, but not marA or rob, restored the parental growth rate in Zn-depleted medium, while deletion of only soxS from the parental strain led to low growth in Zn-depleted medium. Both results suggested that SoxS is a major regulator responsible for growth under Zn-depleted conditions. Gel shift experiments failed to show direct binding of SoxS to the znuCB promoter, thus suggesting indirect control of znuCB expression by SoxS. While SoxS expression in the triple mutant fully restored persistence, increased expression of znuACB via a plasmid in this mutant only partially restored wild-type levels of persistence in the kidney. This work implicates SoxS control of znuCB expression as a key factor in persistence of E. coli in murine pyelonephritis.

Introductory students in a large, multisection course-based undergraduate research experience acquired content knowledge and research skills while studying the evolutionary conservation of enzymes involved in methionine synthesis. The course research project is designed to be sustainable and easily adapted to other research questions and institutional settings.

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