ThefruRBAOperon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood

Abstract

c Bacterial pathogens rely on the availability of nutrients for survival in the host environment. The phosphoenolpyruvate-phosphotransferase system (PTS) is a global regulatory network connecting sugar uptake with signal transduction. Since the fructose PTS has been shown to impact virulence in several streptococci, including the human pathogen Streptococcus pyogenes (the group A Streptococcus [GAS]), we characterized its role in carbon metabolism and pathogenesis in the M1T1 strain 5448. Growth in fructose as a sole carbon source resulted in 103 genes affected transcriptionally, where the fru locus (fruRBA) was the most induced. Reverse transcriptase PCR showed that fruRBA formed an operon which was repressed by FruR in the absence of fructose, in addition to being under carbon catabolic repression. Growth assays and carbon utilization profiles revealed that although the entire fru operon was required for growth in fructose, FruA was the main transporter for fructose and also was involved in the utilization of three additional PTS sugars: cellobiose, mannitol, and N-acetyl-D-galactosamine. The inactivation of sloR, a fruA homolog that also was upregulated in the presence of fructose, failed to reveal a role as a secondary fructose transporter. Whereas the ability of both ⌬fruR and ⌬fruB mutants to survive in the presence of whole human blood or neutrophils was impaired, the phenotype was not reproduced in murine whole blood, and those mutants were not attenuated in a mouse intraperitoneal infection. Since the ⌬fruA mutant exhibited no phenotype in the human or mouse assays, we propose that FruR and FruB are important for GAS survival in a human-specific environment. Bacterial pathogenesis is intimately linked to the availability of nutrients that a pathogen encounters in the host, such as preferred carbohydrate sources for carbon and energy. This paradigm holds true for Gram-positive pathogens in the phylum Firmicutes, where low glucose availability conveys methicillin resistance in Staphylococcus aureus, promotes successful colonization in Streptococcus pneumoniae, stimulates host cell invasion in Listeria monocytogenes, and increases toxin production in Clostridium perfringens (1). These pathogens, like all bacteria, rely on global regulatory networks and dedicated sugar transporters in order to detect the presence of preferred carbon sources as a reflection of the nutrient status of the host environment. This allows for the appropriate coordination of virulence gene expression and disease manifestations in response to surrounding conditions. The phosphoenolpyruvate (PEP)-phosphotransferase system (PTS) is the main system that is utilized by bacteria for the uptake of sugar and sugar derivatives as well as for signal transduction (2). The PTS is made up of distinct proteins, including two cytosolic components, enzyme I (EI) (ptsI) and Hpr (ptsH), and several membrane-bound sugar-specific enzyme IIs (EIIs). Each EII is composed of two cytosolic components (EIIA-B), an integral membrane domain (EIIC), and, in s...

Coordinated Regulation of the EII Man and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways

“…There were also about 70 hypothetical or unknown ORFs that showed reduced expression. Loss of FruK led to increased expression in genes that are likely involved in competence and genetic transformation (the precursor of the competence-stimulating peptide comC, the competence-specific sigma factor comX, and late-competence genes comFA, comFC, comEA, comEC, and the comY operon SMU.1980cϳ1987) (41), DNA repair (3 genes), and bacteriocin production and immunity (12 genes, including those for mutacins IV, V, VI, and TnSmu2) (42, 43), 14 genes that are involved in energy metabolism, including a likely alcohol-acetaldehyde dehydrogenase gene, adhE (SMU.148), that was identified in a study in GAS (29), the glycogen biosynthetic cluster (glg; SMU.1536ϳ1539) required for producing intracellular polysaccharides (44), and 12 more encoding products likely involved in transport.…”

“…A homologous operon in S. gordonii (designated fruRKI or fruRBA) is required for biofilm growth, including growth in a cross-kingdom S. gordonii-Candida albicans biofilm model (27,28). Recently, the homologous fruRBA operon of S. pyogenes was shown to be required for optimal resistance to killing by neutrophils in human blood (29). Collectively, these studies indicate that fructose metabolism via the FruRKI pathway is well conserved in LAB and can contribute to the pathophysiology of these bacteria.…”

The Transcriptional Regulator CpsY Is Important for Innate Immune Evasion in Streptococcus pyogenes

“…Furthermore, there are other instances of GAS regulators that influence resistance to host immunity in a host-specific manner. FruR, the transcriptional repressor of the fruRBA operon encoding the fructose-specific EII of the GAS phosphoenolpyruvate phosphotransferase system (PTS), was shown to mediate innate immune evasion in human host blood but not in murine models of infection (22). Also, the human-specific antimicrobial cathelicidin LL-37 stimulates the CovRS/CsrRS twocomponent regulatory system-dependent expression of the capsule synthesis operon (hasABC), the interleukin 8 (IL-8) protease PrtS/ScpC, and the integrin-like/IgG protease Mac/IdeS (35).…”

“…Total RNA was isolated from GAS grown in enriched medium (THY) to early and mid-and late-logarithmic phase or to mid-logarithmic phase in THY (Klett value, 55 to 65), resuspended in RPMI 1640 -2.05 mM L-glutamine plus 20% plasma isolated from heparinized whole blood from human donors and incubated for 2 h as described above. RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research) with a modified procedure to improve GAS cell disruption, as described previously (22). Briefly, cells were resuspended in 700 l of TRIzol supplemented with ca.…”

“…For example, arginine catabolism through CodYmediated control of the arcABC operon (20) contributes to resistance to inducible nitric oxide synthase (iNOS)-mediated production of nitric oxide (21). Access to carbon sources directly impacts immune evasion, as recently demonstrated by loss of the fruB-encoded 1-phosphopfrucokinase adversely affecting GAS survival in human blood and resistance to phagocytes (22). Thus, it is important to understand how GAS adapts to specific host niches, metabolically and through immune evasion, to effectively address the rising incidence of invasive GAS strains (reviewed in reference 23).…”