Screening of Streptococcus pneumoniae ABC Transporter Mutants Demonstrates that LivJHMGF, a Branched-Chain Amino Acid ABC Transporter, Is Necessary for Disease Pathogenesis

Centre for Respiratory Research, Department of Medicine, University College Medical School, Rayne Institute, London, United Kingdom.
Infection and immunity (Impact Factor: 3.73). 06/2009; 77(8):3412-23. DOI: 10.1128/IAI.01543-08
Source: PubMed

ABSTRACT Bacterial ABC transporters are an important class of transmembrane transporters that have a wide variety of substrates and are important for the virulence of several bacterial pathogens, including Streptococcus pneumoniae. However, many S. pneumoniae ABC transporters have yet to be investigated for their role in virulence. Using insertional duplication mutagenesis mutants, we investigated the effects on virulence and in vitro growth of disruption of 9 S. pneumoniae ABC transporters. Several were partially attenuated in virulence compared to the wild-type parental strain in mouse models of infection. For one ABC transporter, required for full virulence and termed LivJHMGF due to its similarity to branched-chain amino acid (BCAA) transporters, a deletion mutant (DeltalivHMGF) was constructed to investigate its phenotype in more detail. When tested by competitive infection, the DeltalivHMGF strain had reduced virulence in models of both pneumonia and septicemia but was fully virulent when tested using noncompetitive experiments. The DeltalivHMGF strain had no detectable growth defect in defined or complete laboratory media. Recombinant LivJ, the substrate binding component of the LivJHMGF, was shown by both radioactive binding experiments and tryptophan fluorescence spectroscopy to specifically bind to leucine, isoleucine, and valine, confirming that the LivJHMGF substrates are BCAAs. These data demonstrate a previously unsuspected role for BCAA transport during infection for S. pneumoniae and provide more evidence that functioning ABC transporters are required for the full virulence of bacterial pathogens.

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Available from: Jose Yuste, Sep 23, 2014
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    • "There were no significant differences in the bacterial loads between the wild-type and ΔmetQ strains in the BALF, lungs and spleens. Hence although previously we have shown disruption of metQ strain reduced virulence in a competitive model of lung infection [7], the ΔmetQ strain was still able to cause significant infection when given as a pure inoculum. The severe growth defect of the double mutant ΔmetEF/ΔmetQ in human blood prompted us to investigate its growth in a mouse sepsis model. "
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    ABSTRACT: Bacterial pathogens need to acquire nutrients from the host, but for many nutrients their importance during infection remain poorly understood. We have investigated the importance of methionine acquisition and synthesis for Streptococcus pneumoniae growth and virulence using strains with gene deletions affecting a putative methionine ABC transporter lipoprotein (Sp_0149, metQ) and/or methionine biosynthesis enzymes (Sp_0585 - Sp_0586, metE and metF). Immunoblot analysis confirmed MetQ was a lipoprotein and present in all S. pneumoniae strains investigated. However, vaccination with MetQ did not prevent fatal S. pneumoniae infection in mice despite stimulating a strong specific IgG response. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry demonstrated that MetQ has both a high affinity and specificity for L-methionine with a K(D) of ∼25 nM, and a ΔmetQ strain had reduced uptake of C(14)-methionine. Growth of the ΔmetQ/ΔmetEF strain was greatly impaired in chemically defined medium containing low concentrations of methionine and in blood but was partially restored by addition of high concentrations of exogenous methionine. Mixed infection models showed no attenuation of the ΔmetQ, ΔmetEF and ΔmetQ/ΔmetEF strains in their ability to colonise the mouse nasopharnyx. In a mouse model of systemic infection although significant infection was established in all mice, there were reduced spleen bacterial CFU after infection with the ΔmetQ/ΔmetEF strain compared to the wild-type strain. These data demonstrate that Sp_0149 encodes a high affinity methionine ABC transporter lipoprotein and that Sp_0585 - Sp_0586 are likely to be required for methionine synthesis. Although Sp_0149 and Sp_0585-Sp_0586 make a contribution towards full virulence, neither was essential for S. pneumoniae survival during infection.
    PLoS ONE 01/2013; 8(1):e49638. DOI:10.1371/journal.pone.0049638 · 3.23 Impact Factor
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    • "The fifth gene, a putative single-strand DNA-binding protein, has significant sequence similarity to a Salmonella enterica ssDNA-binding protein involved in the regulation of recombination. In addition to the established virulence factors, there was also a putative ATP-binding cassette (ABC) transporter protein, which has been shown to be important in the virulence of other streptococcal bacteria [47], [48]. "
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    ABSTRACT: Streptococcus pyogenes, is an important human pathogen classified within the pyogenic group of streptococci, exclusively adapted to the human host. Our goal was to employ a comparative evolutionary approach to better understand the genomic events concomitant with S. pyogenes human adaptation. As part of ascertaining these events, we sequenced the genome of one of the potential sister species, the agricultural pathogen S. canis, and combined it in a comparative genomics reconciliation analysis with two other closely related species, Streptococcus dysgalactiae and Streptococcus equi, to determine the genes that were gained and lost during S. pyogenes evolution. Genome wide phylogenetic analyses involving 15 Streptococcus species provided convincing support for a clade of S. equi, S. pyogenes, S. dysgalactiae, and S. canis and suggested that the most likely S. pyogenes sister species was S. dysgalactiae. The reconciliation analysis identified 113 genes that were gained on the lineage leading to S. pyogenes. Almost half (46%) of these gained genes were phage associated and 14 showed significant matches to experimentally verified bacteria virulence factors. Subsequent to the origin of S. pyogenes, over half of the phage associated genes were involved in 90 different LGT events, mostly involving different strains of S. pyogenes, but with a high proportion involving the horse specific pathogen S. equi subsp. equi, with the directionality almost exclusively (86%) in the S. pyogenes to S. equi direction. Streptococcus agalactiae appears to have played an important role in the evolution of S. pyogenes with a high proportion of LGTs originating from this species. Overall the analysis suggests that S. pyogenes adaptation to the human host was achieved in part by (i) the integration of new virulence factors (e.g. speB, and the sal locus) and (ii) the construction of new regulation networks (e.g. rgg, and to some extent speB).
    PLoS ONE 05/2012; 7(5):e37607. DOI:10.1371/journal.pone.0037607 · 3.23 Impact Factor
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    • "To obtain nasal washes the exposed trachea was flushed caudally with 200 ␮l PBS and the fluid exiting the nares collected. For the pneumonia challenge, 10 7 cfu S. pneumonia D39 in 50 ␮l PBS was instilled into the nares under deep general halothane anaesthesia 28 days after the final colonising dose [5] [15] [16]. Animals were culled by exsanguination from the femoral artery under pentobarbital anaesthesia. "
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    ABSTRACT: Live attenuated vaccines have been proposed as a strategy to induce protective immunity against infectious diseases. Recent data have demonstrated that nasopharyngeal colonisation with Streptococcus pneumoniae induces protective immunity against subsequent invasive infection, suggesting nasal vaccination with live attenuated bacteria could be a preventative strategy. However the bacterial factors affecting the strength of this adaptive immune response remain unclear. In a direct comparison with the parent wild-type strain, we found that colonisation with bacteria lacking either capsule or surface lipoproteins led to significantly diminished protection. Immunity after colonisation was not dependent on serum IgG to capsular antigens. Colonisation density and duration was reduced for all the non-protective strains, suggesting that protective immunity maybe related to the extent of nasopharyngeal bacterial exposure. To investigate this hypothesis, we utilised an auxotrophic bacterial Δpab strain where duration of colonisation could be controlled by supply and removal of para-amino-benzoic acid (PABA) to mouse drinking water. Supporting colonisation with the Δpab strain for 5 days with PABA led to a faster serum antibody response compared to colonisation for less than 48 h. This enhanced immunogenicity was associated with a trend towards protection. The data presented here aid our understanding of why only certain live attenuated strains are able to function as effective vaccines, and may be valuable in informing the constituents of future live attenuated vaccines.
    Vaccine 05/2012; 30(30):4453-9. DOI:10.1016/j.vaccine.2012.04.080 · 3.62 Impact Factor
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