Streptococcus pyogenes, a multiple-auxotrophic human pathogen, regulates virulence gene expression according to nutritional availability during various stages in the infection process or in different infection sites. We discovered that CvfA influenced the expression of virulence genes according to growth phase and nutritional status. The influence of CvfA in C medium, rich in peptides and poor in carbohydrates, was most pronounced at the stationary phase. Under these conditions, up to 30% of the transcriptome exhibited altered expression; the levels of expression of multiple virulence genes were altered, including the genes encoding streptokinase, CAMP factor, streptolysin O, M protein (more abundant in the CvfA ؊ mutant), SpeB, mitogenic factor, and streptolysin S (less abundant). The increase of carbohydrates or peptides in media restored the levels of expression of the virulence genes in the CvfA ؊ mutant to wild-type levels (emm, ska, and cfa by carbohydrates; speB by peptides). Even though the regulation of gene expression dependent on nutritional stress is commonly linked to the stringent response, the levels of ppGpp were not altered by deletion of cvfA. Instead, CvfA interacted with enolase, implying that CvfA, a putative RNase, controls the transcript decay rates of virulence factors or their regulators according to nutritional status. The virulence of CvfA ؊ mutants was highly attenuated in murine models, indicating that CvfA-mediated gene regulation is necessary for the pathogenesis of S. pyogenes. Taken together, the CvfA-enolase complex in S. pyogenes is involved in the regulation of virulence gene expression by controlling RNA degradation according to nutritional stress.

Small cyclic nucleotide derivatives are employed as second messengers by both prokaryotes and eukaryotes to regulate diverse cellular processes responding to various signals. In bacteria, c-di-AMP has been discovered most recently, and some Gram-positive pathogens including S. pyogenes use this cyclic nucleotide derivative as a second messenger instead of c-di-GMP, a well-studied important bacterial second messenger. GdpP, c-di-AMP phosphodiesterase, is responsible for degrading c-di-AMP inside cells, and the cellular role of GdpP in S. pyogenes has not been examined yet. To test the cellular role of GdpP, we created a strain with a nonpolar inframe deletion of the gdpP gene, and examined the properties of the strain including virulence. From this study, we demonstrated that GdpP influences the biogenesis of SpeB, the major secreted cysteine protease, at a post-translational level, susceptibility to the beta lactam antibiotic ampicillin, and is necessary for full virulence in a murine subcutaneous infection model.

The capsule of Streptococcus pyogenes serves as an adhesin as well as an anti-phagocytic factor by binding to CD44 on keratinocytes of the pharyngeal mucosa and the skin, the main entry sites of the pathogen. We discovered that S. pyogenes HSC5 and MGAS315 strains are further thermoregulated for capsule production at a post-transcriptional level in addition to the transcriptional regulation by the CovRS two-component regulatory system. When the transcription of the hasABC capsular biosynthetic locus was de-repressed through mutation of the covRS system, the two strains, which have been used for pathogenesis studies in the laboratory, exhibited markedly increased capsule production at sub-body temperature. Employing transposon mutagenesis, we found that CvfA, a previously identified membrane-associated endoribonuclease, is required for the thermoregulation of capsule synthesis. The mutation of the cvfA gene conferred increased capsule production regardless of temperature. However, the amount of the capsule transcript was not changed by the mutation, indicating that a post-transcriptional regulator mediates between CvfA and thermoregulated capsule production. When we tested naturally occurring invasive mucoid strains, a high percentage (11/53, 21%) of the strains exhibited thermoregulated capsule production. As expected, the mucoid phenotype of these strains at sub-body temperature was due to mutations within the chromosomal covRS genes. Capsule thermoregulation that exhibits high capsule production at lower temperatures that occur on the skin or mucosal surface potentially confers better capability of adhesion and invasion when S. pyogenes penetrates the epithelial surface.

Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) is the sole enzyme responsible for generation of 5-methyltetrahydrofolate, which is required for methionine synthesis and provision of methyl groups via S-adenosylmethionine. Genome analysis showed that Leishmania species, unlike Trypanosoma brucei and Trypanosoma cruzi, contain genes encoding MTHFR and two distinct methionine synthases. Leishmania MTHFR differed from those in other eukaryotes by the absence of a C-terminal regulatory domain. L. major MTHFR was expressed in yeast and recombinant enzyme was produced in Escherichia coli. MTHFR was not inhibited by S-adenosylmethionine and, uniquely among folate-metabolizing enzymes, showed dual-cofactor specificity with NADH and NADPH under physiological conditions. MTHFR null mutants (mthfr ؊ ) lacked 5-methyltetrahydrofolate, the most abundant intracellular folate, and could not utilize exogenous homocysteine for growth. Under conditions of methionine limitation mthfr ؊ mutant cells grew poorly, whereas their growth was normal in standard culture media. Neither in vitro MTHFR activity nor the growth of mthfr ؊ mutants or MTHFR overexpressors were differentially affected by antifolates known to inhibit parasite growth via targets beyond dihydrofolate reductase and pteridine reductase 1. In a mouse model of infection mthfr ؊ mutants showed good infectivity and virulence, indicating that sufficient methionine is available within the parasitophorous vacuole to meet the needs of the parasite.

Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H4B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H4B levels. Here we explored the consequences of genetic manipulation of the sole L. major phenylalanine hydroxylase (PAH) to explore whether it could account for the Leishmania H4B requirement. L. major PAH resembles AAAHs of other organisms, bearing eukaryotic-type domain organization, and conservation of key catalytic residues including those implicated in pteridine binding. A pah− null mutant and an episomal complemented overexpressing derivative (pah−/+PAH) were readily obtained, and metabolic labeling studies established that PAH was required to hydroxylate Phe to Tyr. Neither WT nor overexpressing lines were able to hydroxylate radiolabeled tyrosine or tryptophan, nor to synthesize catecholamines. WT but not pah− parasites showed reactivity with an antibody to melanin when grown with L-3,4-dihydroxyphenylalanine (L-DOPA), although the reactive product is unlikely to be melanin sensu strictu. WT was auxotrophic for Phe, Trp and Tyr, suggesting that PAH activity was insufficient to meet normal Tyr requirements. However, pah− showed an increased sensitivity to Tyr deprivation, while the pah−/+PAH overexpressor showed increased survival and could be adapted to grow well without added Tyr. pah− showed no alterations in H4B-dependent differentiation, as established by in vitro metacyclogenesis, or survival in mouse or macrophage infections. Thus Leishmania PAH may mitigate but not alleviate Tyr auxotrophy, but plays no essential role in the steps of the parasite infectious cycle. These findings suggest PAH is unlikely to explain the Leishmania requirement for biopterin.