The Metal Homeostasis Protein, Lsp, of Streptococcus pyogenes Is Necessary for Acquisition of Zinc and Virulence

Abstract

"Cluster 9" family lipoproteins function as ligand-binding subunits of ABC-type transporters in maintaining transition metal homeostasis and have been implicated in the virulence of several bacteria. While these proteins share high similarity, the specific metal that they recognize and whether their role in virulence directly involves metal homeostasis cannot be reliably predicted. We examined the cluster 9 protein Lsp of Streptococcus pyogenes and found that specific deletion of lsp produced mutants highly attenuated in a murine model of soft tissue infection. Under standard in vitro conditions, growth of the Lsp ؊ mutant was indistinguishable from that of the wild type, but growth was defective under zinc-limited conditions. The growth defect could be complemented by plasmids expressing wild-type Lsp but not Lsp engineered to lack its putative lipidation residue. Furthermore, Zn 2؉ but not Mn 2؉ rescued Lsp ؊ growth, implicating Zn 2؉ as the physiological ligand for Lsp. Mutation of residues in the putative Zn 2؉ -binding pocket generated variants both hypo-and hyperresistant to zinc starvation, and both mutant classes displayed attenuated virulence. Together, these data suggest that Lsp is a ligand-binding component of an ABC-type zinc permease and that perturbation of zinc homeostasis inhibits the ability of S. pyogenes to cause disease in a zinc-limited host milieu.Transition metals, including iron, zinc, manganese, nickel, and copper, participate in many structural and catalytic functions that are necessary to support the pathogenesis of bacterial infections. However, the ease with which these metals promote cellular electron trafficking also gives them potential to engage in destructive metal-based reactions (25, 59). Thus, a successful pathogen must evolve mechanisms to control the availability and distribution of individual metals within the bacterial cell while exposed to a dynamic host environment with profoundly fluctuating transition metal abundance (13,15,41,62).Evidence is emerging to suggest that the mechanisms by which Streptococcus pyogenes (group A streptococcus) interacts with transition metals play an important role in its ability to cause disease. This gram-positive bacterium is the causative agent of numerous diseases of soft tissue, ranging from selflimiting (e.g., pharyngitis) to destructive and life-threatening (e.g., necrotizing fasciitis), as well as serious postinfectious sequelae such as rheumatic fever and acute glomerulonephritis. A number of metal transporters of S. pyogenes have been characterized, and several of these have been shown to play roles in the virulence of streptococcal infection in various animal models (3,4,40,41,57,70). However, the full repertoire of metal transporters and regulatory elements that comprise the metalloregulatory network of S. pyogenes has not been characterized. In particular, the import and export proteins specific for each of the important transition metals have not been identified. Consequently, global understanding of S. pyogenes transition me...

Znu Is the Predominant Zinc Importer in Yersinia pestis during In Vitro Growth but Is Not Essential for Virulence

“…In contrast to our findings with Y. pestis, mutation of znu ABC transporters in Brucella abortus, Campylobacter jejuni, Haemophilus ducreyi, Pasteurella multocida, Salmonella enterica serovar Typhimurium, and Streptococcus pyogenes causes a loss of virulence or host colonization (1,15,24,42,57,64,67,104,107). The large differences observed between parental and mutant strains suggest that these bacteria may differ from Y. pestis in that they lack a second high-affinity Zn transporter effective in host environments.…”

“…In recent years, the virulence of a variety of bacterial pathogens has been shown to be dependent upon Zn acquisition from the host via Znu or related C9 ABC transporters (1,15,24,42,57,64,67,74,90,104,107). Experiments were undertaken, therefore, to determine whether the Znu system is required for the virulence of Y. pestis.…”

“…Although most SBPs appear to have specificity for a single metal ligand, there is in vitro and in vivo experimental evidence that some non-Znu C9 SBPs possess specificities for multiple metals. The relevance of Zn uptake and the Znu system in particular is highlighted by a number of reports demonstrating that pathogens lacking this transporter are markedly attenuated in virulence (1,15,24,42,57,64,67,74,90,104,107).…”

Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence

“…However, in many low-GC Gram-positive pathogens, mutations in either lgt or lsp also led to impaired pathogenicity. This was the case in various pathogenic streptococcal species such as Streptococcus pneumoniae (21,22), Streptococcus agalactiae (23,24), Streptococcus pyogenes, and Streptococcus equi (25,26). In Listeria monocytogenes, the lgt mutant was impaired in invasion and intracellular survival, was markedly attenuated in a mouse infection model, was unable to induce TLR2-dependent activation of NF-B, and exhibited increased susceptibility to cationic peptides (27,28).…”

Copper Tolerance and Characterization of a Copper-Responsive Operon, copYAZ , in an M1T1 Clinical Strain of Streptococcus pyogenes

“…Homeostatic systems involved in regulating intracellular levels of iron, manganese, zinc, cobalt, and heme play a critical role in S. pyogenes survival, as demonstrated in a wide variety of animal models (5)(6)(7). However, the mechanism(s) that prevents copper toxicity in S. pyogenes has not been previously described.…”