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

Basavanna, Shilpa; Khandavilli, Suneeta; Yuste, José; Cohen, Jonathan; Hosie, Arthur H.F.; Webb, Alexander J.; Thomas, Gavin H.; Brown, Jeremy S.

doi:10.1128/iai.01543-08

Cited by 82 publications

(114 citation statements)

References 44 publications

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“…Indeed, pneumococci possess multiple mechanisms for carbohydrate uptake and metabolism that are intricately linked to their pathogenesis (Iyer et al, 2005;Iyer & Camilli, 2007). Transport and biosynthesis of amino acids, manganese and iron have been shown to be important for pneumococcal pathogenesis (Basavanna et al, 2009;Gupta et al, 2009;Nanduri et al, 2008;Rosch et al, 2009;Yesilkaya et al, 2000). A thorough understanding of pneumococcal metabolism is required for designing effective therapeutic and prophylactic strategies.…”

Section: Introductionmentioning

confidence: 99%

Polyamine biosynthesis and transport mechanisms are crucial for fitness and pathogenesis of Streptococcus pneumoniae

et al. 2011

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Polyamines such as cadaverine, putrescine and spermidine are polycationic molecules that have pleiotropic effects on cells via their interaction with nucleic acids. Streptococcus pneumoniae (the pneumococcus) is a Gram-positive pathogen capable of causing pneumonia, septicaemia, otitis media and meningitis. Pneumococci have a polyamine transport operon (potABCD) responsible for the binding and transport of putrescine and spermidine, and can synthesize cadaverine and spermidine using their lysine decarboxylase (cad) and spermidine synthase (speE) enzymes. Previous studies from our laboratory have shown that an increase in PotD expression is seen following exposure to various stresses, while during infection, potD inactivation significantly attenuates pneumococcal virulence, and anti-PotD immune responses are protective in mice. In spite of their relative importance, not much is known about the global contribution of polyamine biosynthesis and transport pathways to pneumococcal disease. Mutants deficient in polyamine biosynthesis (DspeE or Dcad) or transport genes (DpotABCD) were constructed and were found to be attenuated in murine models of pneumococcal colonization and pneumonia, either alone or in competition with the wild-type strain. The DspeE mutant was also attenuated during invasive disease, while the potABCD and cad genes seemed to be dispensable. HPLC analyses showed reduced intracellular polyamine levels in all mutant strains compared with wild-type bacteria. Highthroughput proteomic analyses indicated reduced expression of growth, replication and virulence factors in mutant strains. Thus, polyamine biosynthesis and transport mechanisms are intricately linked to the fitness, survival and pathogenesis of the pneumococcus in host microenvironments, and may represent important targets for prophylactic and therapeutic interventions.

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Section: Introductionmentioning

confidence: 99%

Polyamine biosynthesis and transport mechanisms are crucial for fitness and pathogenesis of Streptococcus pneumoniae

et al. 2011

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“…The transport mechanisms described for acquisition of the BCAAs in bacteria include secondary transporters belonging to the Leu, Ile, Val:cation symporter (LIVCS) family (e.g., BrnQ, BraB, BraZ, and BcaP) (4,(6)(7)(8)(9) and the LIV-I ABC transporter (10)(11)(12)(13). With the exception of an ABC transporter in Streptococcus pneumoniae (14), secondary transport, which couples the movement of an ion, usually Na ϩ or H ϩ , down its concentration gradient with the movement of another molecule against its concentration gradient, is the primary means of BCAA acquisition in Gram-positive bacteria.…”

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confidence: 99%

Role of BrnQ1 and BrnQ2 in Branched-Chain Amino Acid Transport and Virulence in Staphylococcus aureus

et al. 2015

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The branched-chain amino acids (BCAAs; Ile, Leu, and Val) not only are important nutrients for the growth of Staphylococcus aureus but also are corepressors for CodY, which regulates virulence gene expression, implicating BCAAs as an important link between the metabolic state of the cell and virulence. BCAAs are either synthesized intracellularly or acquired from the environment. S. aureus encodes three putative BCAA transporters, designated BrnQ1, BrnQ2, and BrnQ3; their functions have not yet been formally tested. In this study, we mutated all three brnQ paralogs so as to characterize their substrate specificities and their roles in growth in vitro and in vivo. We demonstrated that in the community-associated, methicillin-resistant S. aureus (CA-MRSA) strain USA300, BrnQ1 is involved in uptake of all three BCAAs, BrnQ2 transports Ile, and BrnQ3 does not have a significant role in BCAA transport under the conditions tested. Of the three, only BrnQ1 is essential for USA300 to grow in a chemically defined medium that is limited for Leu or Val. Interestingly, we observed that a brnQ2 mutant grew better than USA300 in media limited for Leu and Val, owing to the fact that this mutation leads to overexpression of brnQ1. In a murine infection model, the brnQ1 mutant was attenuated, but in contrast, brnQ2 mutants had significantly increased virulence compared to that of USA300, a phenotype we suggest is at least partially linked to enhanced in vivo scavenging of Leu and Val through BrnQ1. These data uncover a hitherto-undiscovered connection between nutrient acquisition and virulence in CA-MRSA. Staphylococcus aureus is a highly successful human pathogen that succeeds at infecting virtually every body site, causing skin, soft tissue, respiratory, bone, joint, and endovascular infections (1). Maintenance of metabolic homeostasis is important for its infection process, as the majority of genes necessary for infection identified in large-scale signature-tagged mutagenesis (STM) screens fall into the categories of metabolism, transport, and biosynthesis (2, 3). Acquisition of host-derived nutrients, specifically amino acids, appears to be an important mechanism of meeting nutritional needs, as a large proportion of attenuated STM strains contain mutations in amino acid transporters (3).Amino acid transporters are ubiquitous in bacteria and are typically selective for transporting either one amino acid or several amino acids with related structures (4). The branched-chain amino acids (BCAAs; Ile, Leu, and Val) are hydrophobic amino acids typically found in the core in globular proteins or in the transmembrane domain in cell surface proteins (5). The transport mechanisms described for acquisition of the BCAAs in bacteria include secondary transporters belonging to the Leu, Ile, Val:cation symporter (LIVCS) family (e.g., BrnQ, BraB, BraZ, and BcaP) (4, 6-9) and the LIV-I ABC transporter (10-13). With the exception of an ABC transporter in Streptococcus pneumoniae (14), secondary transport, which couples the movement of an ion...

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“…Filters were then placed in scintillation vials and radioactivity was counted in a Wallac model 1409 liquid scintillation counter. Glucose (D- [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]-glucose; ARC 0120A) and raffinose ([galactose-6-imal medium, as previously described (41). Cells (1.5-ml aliquots) were then exposed to 130 l of either PBS alone or PBS containing glucose, sucrose, or lactose to a final concentration of 20 mM.…”

Section: Rna Isolation and Quantitative Rt-pcr (Qrt-pcr)mentioning

confidence: 99%

“…RafK-deficient pneumococcus are attenuated in virulence in mice. Several ABC transporters have been identified as pneumococcal virulence factors, with some transporters being both necessary for uptake of nutrients and also involved in attachment to various ligands that could help the bacteria colonize and invade its host (4,8,13,18,22,30). As DLDH-negative pneumococci have a striking avirulent phenotype in murine colonization and infection models, we were interested in better understanding the contribution of the raffinose transport system to this avirulent phenotype.…”

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confidence: 99%

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Role of Dihydrolipoamide Dehydrogenase in Regulation of Raffinose Transport in Streptococcus pneumoniae

Tyx

Håkansson

2011

J Bacteriol

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Streptococcus pneumoniae strains lacking the enzyme dihydrolipoamide dehydrogenase (DLDH) show markedly reduced ability to grow on raffinose and stachyose as sole carbon sources. Import of these sugars occurs through the previously characterized raffinose ATP-binding cassette (ABC) transport system, encoded by the raf operon, that lacks the necessary ATP-binding protein. In this study, we identified the raffinose ATP-binding protein RafK and showed that it was directly involved in raffinose and stachyose import. RafK carries a C-terminal regulatory domain present in a subset of ATP-binding proteins that has been involved in both direct regulation of transporter activity (inducer exclusion) and transcription of transporter genes. Pneumococci lacking RafK showed a 50-to 80-fold reduction in expression of the raf operon genes aga (alpha-galactosidase) and rafEFG (raffinose substrate binding and permease genes), and both glucose and sucrose inhibited raffinose uptake through inducer exclusion. Like RafK, the presence of DLDH also activated the expression of raf operon genes, as DLDH-negative pneumococci showed a significantly decreased expression of aga and rafEFG, but DLDH did not regulate rafK or the putative regulatory genes rafR and rafS. DLDH also bound directly to RafK both in vitro and in vivo, indicating the possibility that DLDH regulates raffinose transport by a direct interaction with the regulatory domain of the transporter. Finally, although not as attenuated as DLDHnegative bacteria, pneumococci lacking RafK were significantly outcompeted by wild-type bacteria in colonization experiments of murine lung and nasopharynx, indicating a role for raffinose and stachyose transport in vivo.Dihydrolipoamide dehydrogenases (DLDH) are enzymes classically involved in the reoxidation of dihydrolipoamide during conversion of 2-oxo acids (such as pyruvate) in several multienzyme complexes in the central metabolism (34, 52). However, previous work from our and another laboratory have shown that DLDH is not involved in metabolizing 2-oxo acids in the pneumococcus, indicating that DLDH must serve other functions (41,42). This is supported by evidence that organisms that lack 2-oxo acid dehydrogenases still express a DLDH protein (9, 10). One such function was suggested by Richarme in the 1980s when he showed that Escherichia coli strains that fail to make lipoic acid or strains treated with an inhibitor that mainly inhibits DLDH function resulted in reduced import of galactose, maltose, and ribose through ATP-binding cassette (ABC) transporters (35). We then showed that inactivation of DLDH in the pneumococcus results in an inability of the bacteria to import and utilize galactose and the alpha-galactoside sugars raffinose and stachyose and that a lack of DLDH is associated with an almost complete attenuation of this strain in animal infection experiments (41).Transport of carbohydrates and other energy sources is important for many aspects of bacterial life and therefore highly regulated. Fitness in the bacterial host ...

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

Cited by 82 publications

References 44 publications

Polyamine biosynthesis and transport mechanisms are crucial for fitness and pathogenesis of Streptococcus pneumoniae

Polyamine biosynthesis and transport mechanisms are crucial for fitness and pathogenesis of Streptococcus pneumoniae

Role of BrnQ1 and BrnQ2 in Branched-Chain Amino Acid Transport and Virulence in Staphylococcus aureus

Role of Dihydrolipoamide Dehydrogenase in Regulation of Raffinose Transport in Streptococcus pneumoniae

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