A proteomic analysis of penicillin resistance in <i>Streptococcus pneumoniae</i> reveals a novel role for PstS, a subunit of the phosphate ABC transporter

Soualhine, Hafid; Brochu, Vicky; Ménard, F.; Papadopoulou, Barbara; Weiss, Karl Heinz; Bergeron, Michel G.; Légaré, Danielle; Drummelsmith, Jolyne; Ouellette, Marc

doi:10.1111/j.1365-2958.2005.04914.x

Cited by 49 publications

(45 citation statements)

References 47 publications

(60 reference statements)

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“…Cultures were incubated for 16-24 h in a 5% CO 2 atmosphere at 37°C. In order to select resistant cells, we subcultured R6 or CCRI 1974 in medium containing increasing concentration gradients of linezolid as described previously for other antibiotics (Martineau et al 2000;Soualhine et al 2005). Mutants were selected in a stepwise fashion by picking colonies growing at the highest antibiotic concentration at each stage.…”

Section: Methodsmentioning

confidence: 99%

“…For episomal expression, the entire coding sequences of genes of interest were PCR amplified with primers containing BamHI and XbaI and cloned into the S. pneumoniae-E. coli shuttle vector pDL289 (Buckley et al 1995), a kind gift of D. Cvitkovitch (University of Toronto). The plasmid DNAs (2 mg) were introduced in S. pneumoniae in which competence was induced using a competence peptide as described (Soualhine et al 2005). Transformants were selected in media containing 500 mg/ mL kanamycin.…”

Section: Dna Constructsmentioning

confidence: 99%

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Genome sequencing of linezolid-resistant Streptococcus pneumoniae mutants reveals novel mechanisms of resistance

Feng¹,

Lupien²,

Gingras³

et al. 2009

Genome Res.

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103

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Linezolid is a member of a novel class of antibiotics, with resistance already being reported. We used whole-genome sequencing on three independent Streptococcus pneumoniae strains made resistant to linezolid in vitro in a step-by-step fashion. Analysis of the genome assemblies revealed mutations in the 23S rRNA gene in all mutants including, notably, G2576T, a previously recognized resistance mutation. Mutations in an additional 31 genes were also found in at least one of the three sequenced genomes. We concentrated on three new mutations that were found in at least two independent mutants. All three mutations were experimentally confirmed to be involved in antibiotic resistance. Mutations upstream of the ABC transporter genes spr1021 and spr1887 were correlated with increased expression of these genes and neighboring genes of the same operon. Gene inactivation supported a role for these ABC transporters in resistance to linezolid and other antibiotics. The hypothetical protein spr0333 contains an RNA methyltransferase domain, and mutations within that domain were found in all S. pneumoniae linezolid-resistant strains. Primer extension experiments indicated that spr0333 methylates G2445 of the 23S rRNA and mutations in spr0333 abolished this methylation. Reintroduction of a nonmutated version of spr0333 in resistant bacteria reestablished G2445 methylation and led to cells being more sensitive to linezolid and other antibiotics. Interestingly, the spr0333 ortholog was also mutated in a linezolid-resistant clinical Staphylococcus aureus isolate. Whole-genome sequencing and comparative analyses of S. pneumoniae resistant isolates was useful for discovering novel resistance mutations.

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Section: Methodsmentioning

confidence: 99%

Section: Dna Constructsmentioning

confidence: 99%

Genome sequencing of linezolid-resistant Streptococcus pneumoniae mutants reveals novel mechanisms of resistance

Feng¹,

Lupien²,

Gingras³

et al. 2009

Genome Res.

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103

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Section: Introductionmentioning

confidence: 99%

“…P. mirabilis HI4320 possesses a pst operon identical in gene organization to that observed in Escherichia coli. Previous studies have suggested that the roles of the Pst transport system in pathogenesis include regulation of invasion (Lucas et al, 2000;Mathew et al, 2001;Sinai & Bavoil, 1993), antibiotic resistance (Soualhine et al, 2005), colonization (Buckles et al, 2006;Daigle et al, 1995;Lamarche et al, 2005;Orihuela et al, 2001;Peirs et al, 2005;Runyen-Janecky et al, 2005), and biofilm formation (Monds et al, 2001).Since (i) earlier work has alluded to the importance of this system during biofilm formation (Monds et al, 2001(Monds et al, , 2007 and (ii) the formation of crystalline biofilms is a characteristic of UTIs associated with P. mirabilis, we hypothesized that the Pst system of P. mirabilis HI4320 is involved in biofilm formation during establishment of a UTI. To assess the role of the Pst system, this study focused upon determining previously undetected in vitro phenotypes of the Dpst mutants by comparing biofilm formation between the wild-type HI4320 and DpstA and DpstS mutants via confocal laser scanning microscopy (CLSM) and quantitative analysis of surface coverage, and by comparing in vitro protein expression of these strains grown planktonically and as a biofilm.…”

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confidence: 99%

The high-affinity phosphate transporter Pst in Proteus mirabilis HI4320 and its importance in biofilm formation

et al. 2009

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Proteus mirabilis causes urinary tract infections (UTIs) in individuals requiring long-term indwelling catheterization. The pathogenesis of this uropathogen is mediated by a number of virulence factors and the formation of crystalline biofilms. In addition, micro-organisms have evolved complex systems for the acquisition of nutrients, including the phosphate-specific transport system, which has been shown to be important in biofilm formation and pathogenesis. A functional Pst system is important during UTIs caused by P. mirabilis HI4320, since transposon mutants in the PstS periplasmic binding protein and the PstA permease protein were attenuated in the CBA mouse model of UTI. These mutants displayed a defect in biofilm formation when grown in human urine. This study focuses on a comparison of the proteomes during biofilm and planktonic growth in phosphate-rich medium and human urine, and microscopic investigations of biofilms formed by the pst mutants. Our data suggest that (i) the Dpst mutants, and particularly the DpstS mutant, are defective in biofilm formation, and (ii) the proteomes of these mutants differ significantly from that of the wild-type. Therefore, since the Pst system of P. mirabilis HI4320 negatively regulates biofilm formation, this system is important for the pathogenesis of these organisms during complicated UTIs. INTRODUCTIONProteus mirabilis -a member of the family Enterobacteriaceae -is the most common aetiological agent responsible for complicated urinary tract infections (UTIs) (Mobley, 1996). Subpopulations at higher risk for infection by this pathogen include those with long-term indwelling catheterization as well as those with structural and functional abnormalities within the urinary tract (Mobley, 1996). Clinical syndromes associated with P. mirabilis include cystitis and pyelonephritis, with possible complications from stone formation and bacteraemia (Mobley, 1996). These micro-organisms survive in the urinary tract by virtue of their production of a battery of virulence factors, including urease, flagella, fimbriae, haemolysin and IgA protease (Belas, 1996;Mobley et al., 1994;Musher et al., 1975;Peerbooms et al., 1984; Walker et al., 1999), and formation of crystalline biofilm on indwelling catheters (Stickler et al., 1993).Biofilm formation -one of the most important mechanisms of pathogenicity of this micro-organism in the urinary tract -may be defined as a community of surface-attached bacteria encased in an extracellular matrix consisting of secreted carbohydrates, proteins and DNA. Indeed, biofilms have been associated with the virulence of a number of pathogens (Castelli et al., 2006). Organisms in these communities display phenotypic differences from planktonic cells, including a slower rate of growth and increased resistance to antibiotics (Lewis, 2001;Stoodley et al., 2002). P. mirabilis biofilms in urine are unique in that the extracellular polysaccharide matrix is enmeshed with struvite and carbonate apatite crystals (Jones et al., 2006).Abbreviations: CLSM, confocal ...

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“…Moreover, ABC transporters together with neighboring two-component systems, which regulate their expression, could coevolve to form self-sufficient detoxification modules against antimicrobial peptides in Firmicutes bacteria (44). A significant correlation between penicillin resistance and upregulation of ABC transporters was also reported (45). These studies indicate that the overexpression of ABC transporters might also be involved in CRO resistance by enhancing the activity of drug transport.…”

Section: Discussionmentioning

confidence: 70%

Novel Genes Related to Ceftriaxone Resistance Found among Ceftriaxone-Resistant Neisseria gonorrhoeae Strains Selected In Vitro

Gong

Lai

Liu

et al. 2016

Antimicrob Agents Chemother

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dThe emergence of ceftriaxone-resistant Neisseria gonorrhoeae is currently a global public health concern. However, the mechanism of ceftriaxone resistance is not yet fully understood. To investigate the potential genes related to ceftriaxone resistance in Neisseria gonorrhoeae, we subcultured six gonococcal strains with increasing concentrations of ceftriaxone and isolated the strains that became resistant. After analyzing several frequently reported genes involved in ceftriaxone resistance, we found only a single mutation in penA (A501V). However, differential analysis of the genomes and transcriptomes between pre-and postselection strains revealed many other mutated genes as well as up-and downregulated genes. Transformation of the mutated penA gene into nonresistant strains increased the MIC between 2.0-and 5.3-fold, and transformation of mutated ftsX increased the MIC between 3.3-and 13.3-fold. Genes encoding the ABC transporters FarB, Tfq, Hfq, and ExbB were overexpressed, while pilM, pilN, and pilQ were downregulated. Furthermore, the resistant strain developed cross-resistance to penicillin and cefuroxime, had an increased biochemical metabolic rate, and presented fitness defects such as prolonged growth time and downregulated PilMNQ. In conclusion, antimicrobial pressure could result in the emergence of ceftriaxone resistance, and the evolution of resistance of Neisseria gonorrhoeae to ceftriaxone is a complicated process at both the pretranscriptional and posttranscriptional levels, involving several resistance mechanisms of increased efflux and decreased entry. Gonorrhea is one of the most prevalent sexually transmitted infections (STIs), and it is associated with many severe clinical symptoms such as prostatitis, seminal vesiculitis, pelvic inflammation, infertility, and ophthalmia neonatorum (1). Although epidemiological data show a decreased trend in gonorrhea incidence as a whole, in some countries, the incidence is increasing, especially in specific population groups (adolescents, men who have sex with men, or sex workers) or specific venues (brothels) (2-5). The pathogen responsible for this disease is Neisseria gonorrhoeae. According to worldwide surveillance reports, N. gonorrhoeae has developed resistance to several kinds of antibiotics over the past decades (6-10). Of increasing concern is the prevalence of ceftriaxone (CRO)-resistant N. gonorrhoeae (11), because CRO is the first-line empirical drug for gonorrhea treatment in dual therapy with azithromycin or doxycycline recommended by the WHO (covering the Western Pacific region and the Southeast Asian region) and the CDC (12-14). Several CRO-resistant gonococcal strains, such as H041 and F89, were isolated recently from patients who had a treatment failure with CRO (H041) and cefixime (F89) (15-18). Highlighting the seriousness of these resistant strains, Ohnishi et al. proposed the concept of an N. gonorrhoeae "superbug" in 2011 (17). Uncovering the mechanisms of CRO resistance is now a critical topic of N. gonorrhoeae research.Previous s...

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A proteomic analysis of penicillin resistance in Streptococcus pneumoniae reveals a novel role for PstS, a subunit of the phosphate ABC transporter

Cited by 49 publications

References 47 publications

Genome sequencing of linezolid-resistant Streptococcus pneumoniae mutants reveals novel mechanisms of resistance

Genome sequencing of linezolid-resistant Streptococcus pneumoniae mutants reveals novel mechanisms of resistance

The high-affinity phosphate transporter Pst in Proteus mirabilis HI4320 and its importance in biofilm formation

Novel Genes Related to Ceftriaxone Resistance Found among Ceftriaxone-Resistant Neisseria gonorrhoeae Strains Selected In Vitro

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