In silico and experimental analysis of the repeated domains in BvaP, a protein important for GBS vaginal colonization.

Streptococcus agalactiae (group B strep, GBS) infections in neonates are often fatal and strongly associated with maternal GBS vaginal colonization. Previously, we highlighted the importance of a formerly uncharacterized protein, BvaP, in GBS vaginal colonization. BvaP is highly conserved across GBS and is made up of repeated domains, with a variable number of repeats between strains. Here, we evaluate the prevalence of BvaP repeated domains and their relevance in phenotypes previously associated with vaginal colonization. Using in silico analysis, we found that the number of repeats in the BvaP protein does not generally appear to be associated with serotype, isolation site, or host. Using BvaP truncations in GBS strain A909, we determined that a smaller number of repeats was correlated with decreased bacterial chain length, but adherence to vaginal epithelial cells was complemented using BvaP containing one, two, three, or five repeats. Future research will be geared toward understanding the host immune response to BvaP in vivo and whether vaginal carriage or host response is dependent on the BvaP repeated domains.

A Novel Conserved Protein in Streptococcus agalactiae, BvaP, Is Important for Vaginal Colonization and Biofilm Formation.

Streptococcus agalactiae (group B streptococcus [GBS]) infections in neonates are often fatal and strongly associated with maternal GBS vaginal colonization. Here, we investigated the role of an uncharacterized protein, BvaP, in GBS vaginal colonization. bvaP was previously identified as the most highly upregulated gene in the GBS A909 transcriptome when comparing vaginal colonization to growth in liquid culture. We found that the absence of BvaP affects the ability of GBS to adhere to extracellular matrix components and human vaginal epithelial cells, and the ability of a ΔbvaP mutant to colonize the murine vaginal tract was significantly decreased. Cellular morphological alterations such as changes in cell shape, chain length, and clumping were also observed in a knockout mutant strain. Given its high expression level in vivo, high degree of conservation among GBS strains, and role in vaginal colonization, BvaP may be an eligible target for GBS vaccination and/or drug therapy. IMPORTANCE Neonatal GBS disease is a major cause of morbidity and mortality, and maternal vaginal colonization is the leading risk factor for the disease. Colonization prevention would greatly impact the rates of disease transmission, but vaccine development has stalled as capsular polysaccharide vaccines have low immunogenicity in vivo. While these vaccines are still in development, the addition of a protein conjugate may prove fruitful in increasing immunogenicity and strain coverage across GBS serotypes. Previous research identified sak_1753 as a highly upregulated gene during murine vaginal colonization. This study reveals that Sak_1753 is required to maintain proper GBS cellular morphology and colonization phenotypes and is required for full in vivo vaginal colonization in a murine model. We have renamed Sak_1753 group B streptococcus vaginal adherence protein (BvaP). The findings of this study indicate that BvaP is important for GBS colonization of the vaginal tract and, given its high expression level in vivo and strain conservation, may be a candidate for vaccine development.

HupZ, a Unique Heme-Binding Protein, Enhances Group A Streptococcus Fitness During Mucosal Colonization.

Group A Streptococcus (GAS) is a major pathogen that causes simple and invasive infections. GAS requires iron for metabolic processes and pathogenesis, and heme is its preferred iron source. We previously described the iron-regulated hupZ in GAS, showing that a recombinant HupZ-His6 protein binds and degrades heme. The His6 tag was later implicated in heme iron coordination by HupZ-His6. Hence, we tested several recombinant HupZ proteins, including a tag-free protein, for heme binding and degradation in vitro. We established that HupZ binds heme but without coordinating the heme iron. Heme-HupZ readily accepted exogenous imidazole as its axial heme ligand, prompting degradation. Furthermore, HupZ bound a fragment of heme c (whose iron is coordinated by the cytochrome histidine residue) and exhibited limited degradation. GAS, however, did not grow on a heme c fragment as an iron source. Heterologous HupZ expression in Lactococcus lactis increased heme b iron use. A GAS hupZ mutant showed reduced growth when using hemoglobin as an iron source, increased sensitivity to heme toxicity, and decreased fitness in a murine model for vaginal colonization. Together, the data demonstrate that HupZ contributes to heme metabolism and host survival, likely as a heme chaperone. HupZ is structurally similar to the recently described heme c-degrading enzyme, Pden_1323, suggesting that the GAS HupZ might be divergent to play a new role in heme metabolism.

A Novel Heme Transporter from the Energy Coupling Factor Family Is Vital for Group A Streptococcus Colonization and Infections.

Group A streptococcus (GAS) produces millions of infections worldwide, including mild mucosal infections, postinfection sequelae, and life-threatening invasive diseases. During infection, GAS readily acquires nutritional iron from host heme and hemoproteins. Here, we identified a new heme importer, named SiaFGH, and investigated its role in GAS pathophysiology. The SiaFGH proteins belong to a group of transporters with an unknown ligand from the recently described family of energy coupling factors (ECFs). A siaFGH deletion mutant exhibited high streptonigrin resistance compared to the parental strain, suggesting that iron ions or an iron complex is the likely ligand. Iron uptake and inductively coupled plasma mass spectrometry (ICP-MS) studies showed that the loss of siaFGH did not impact GAS import of ferric or ferrous iron, but the mutant was impaired in using hemoglobin iron for growth. Analysis of cells growing on hemoglobin iron revealed a substantial decrease in the cellular heme content in the mutant compared to the complemented strain. The induction of the siaFGH genes in trans resulted in the induction of heme uptake. The siaFGH mutant exhibited a significant impairment in murine models of mucosal colonization and systemic infection. Together, the data show that SiaFGH is a new type of heme importer that is key for GAS use of host hemoproteins and that this system is imperative for bacterial colonization and invasive infection.IMPORTANCE ECF systems are new transporters that take up various vitamins, cobalt, or nickel with a high affinity. Here, we establish the GAS SiaFGH proteins as a new ECF module that imports heme and demonstrate its importance in virulence. SiaFGH is the first heme ECF system described in bacteria. We identified homologous systems in the genomes of related pathogens from the Firmicutes phylum. Notably, GAS and other pathogens that use a SiaFGH-type importer rely on host hemoproteins for a source of iron during infection. Hence, recognizing the function of this noncanonical ABC transporter in heme acquisition and the critical role that it plays in disease has broad implications.