Howard F. Jenkinson

Streptococcus Adherence and Colonization

SUMMARY Streptococci readily colonize mucosal tissues in the nasopharynx; the respiratory, gastrointestinal, and genitourinary tracts; and the skin. Each ecological niche presents a series of challenges to successful colonization with which streptococci have to contend. Some species exist in equilibrium with their host, neither stimulating nor submitting to immune defenses mounted against them. Most are either opportunistic or true pathogens responsible for diseases such as pharyngitis, tooth decay, necrotizing fasciitis, infective endocarditis, and meningitis. Part of the success of streptococci as colonizers is attributable to the spectrum of proteins expressed on their surfaces. Adhesins enable interactions with salivary, serum, and extracellular matrix components; host cells; and other microbes. This is the essential first step to colonization, the development of complex communities, and possible invasion of host tissues. The majority of streptococcal adhesins are anchored to the cell wall via a C-terminal LPxTz motif. Other proteins may be surface anchored through N-terminal lipid modifications, while the mechanism of cell wall associations for others remains unclear. Collectively, these surface-bound proteins provide Streptococcus species with a “coat of many colors,” enabling multiple intimate contacts and interplays between the bacterial cell and the host. In vitro and in vivo studies have demonstrated direct roles for many streptococcal adhesins as colonization or virulence factors, making them attractive targets for therapeutic and preventive strategies against streptococcal infections. There is, therefore, much focus on applying increasingly advanced molecular techniques to determine the precise structures and functions of these proteins, and their regulatory pathways, so that more targeted approaches can be developed.

Invasion of dentinal tubules by oral bacteria.

Bacterial invasion of dentinal tubules commonly occurs when dentin is exposed following a breach in the integrity of the overlying enamel or cementum. Bacterial products diffuse through the dentinal tubule toward the pulp and evoke inflammatory changes in the pulpo-dentin complex. These may eliminate the bacterial insult and block the route of infection. Unchecked, invasion results in pulpitis and pulp necrosis, infection of the root canal system, and periapical disease. While several hundred bacterial species are known to inhabit the oral cavity, a relatively small and select group of bacteria is involved in the invasion of dentinal tubules and subsequent infection of the root canal space. Gram-positive organisms dominate the tubule microflora in both carious and non-carious dentin. The relatively high numbers of obligate anaerobes present-such as Eubacterium spp., Propionibacterium spp., Bifidobacterium spp., Peptostreptococcus micros, and Veillonella spp.-suggest that the environment favors growth of these bacteria. Gram-negative obligate anaerobic rods, e.g., Porphyromonas spp., are less frequently recovered. Streptococci are among the most commonly identified bacteria that invade dentin. Recent evidence suggests that streptococci may recognize components present within dentinal tubules, such as collagen type I, which stimulate bacterial adhesion and intra-tubular growth. Specific interactions of other oral bacteria with invading streptococci may then facilitate the invasion of dentin by select bacterial groupings. An understanding the mechanisms involved in dentinal tubule invasion by bacteria should allow for the development of new control strategies, such as inhibitory compounds incorporated into oral health care products or dental materials, which would assist in the practice of endodontics.

Streptococcal Adhesion and Colonization

Streptococci express arrays of adhesins on their cell surfaces that facilitate adherence to substrates present in their natural environment within the mammalian host. A consequence of such promiscuous binding ability is that streptococcal cells may adhere simultaneously to a spectrum of substrates, including salivary glycoproteins, extracellular matrix and serum components, host cells, and other microbial cells. The multiplicity of streptococcal adherence interactions accounts, at least in part, for their success in colonizing the oral and epithelial surfaces of humans. Adhesion facilitates colonization and may be a precursor to tissue invasion and immune modulation, events that presage the development of disease. Many of the streptococcal adhesins and virulence-related factors are cell-wall-associated proteins containing repeated sequence blocks of amino acids. Linear sequences, both within the blocks and within non-repetitive regions of the proteins, have been implicated in substrate binding. Sequences and functions of these proteins among the streptococci have become assorted through gene duplication and horizontal transfer between bacterial populations. Several adhesins identified and characterized through in vitro binding assays have been analyzed for in vivo expression and function by means of animal models used for colonization and virulence. Information on the molecular structure of adhesins as related to their in vivo function will allow for the rational design of novel acellular vaccines, recombinant antibodies, and adhesion agonists for the future control or prevention of streptococcal colonization and streptococcal diseases.

Out of the iron age: new insights into the critical role of manganese homeostasis in bacteria.

Manganese (Mn) is required for the growth and survival of most, if not all, living organisms. Until recently, relatively little was known about how bacteria take up trace nutrients such as Mn, nickel (Ni), copper (Cu) and zinc (Zn), or about how they regulate intracellular levels of these in response to availability and demand. Over the last 5 years a large number of systems involved in these processes have been identified and characterized. This has led to new insights into transition metal ion homeostasis in Gram-positive and Gram-negative bacteria. Metal ions, including Mn, iron (Fe), cobalt (Co), Ni, Cu and Zn, are both essential and potentially toxic. Therefore, homeostatic regulation of their intracellular concentrations is critical.

Structure, function and immunogenicity of streptococcal antigen I/II polypeptides

The antigen I/II family of cell‐surface‐anchored polypeptides in oral streptococci are structurally complex multi‐functional adhesins, with multiple ligand‐binding sites. Discrete regions within these polypeptides bind human salivary glycoproteins, other microbial cells, and calcium. Sequences within the N‐terminal region bind preferentially fluid‐phase glycoproteins, while the C‐terminal half of the polypeptide contains species‐specific adhesion‐mediating sequences that bind surface‐immobilized glycoproteins. These features may assist streptococcal adhesion to oral surface receptors despite the presence of excess fluid‐phase receptors. Immunological studies reveal an array of T‐cell and B‐cell epitopes presented by antigen I/II polypeptides and suggest the occurrence of natural suppression of human antibodies to the adhesion‐mediating sequences. The functional and immunological properties of antigen I/II proteins may account to a major extent for the success of oral streptococci colonizing and surviving within the human host.

The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence.

Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins mediating these reactions are largely uncharacterized. In this report we describe a novel pneumococcal protein PavA, which binds fibronectin and is associated with pneumococcal adhesion and virulence. The pavA gene, present in 64 independent isolates of S. pneumoniae tested, encodes a 551 amino acid residue polypeptide with 67% identical amino acid sequence to Fbp54 protein in Streptococcus pyogenes. PavA localized to the pneumococcal cell outer surface, as demonstrated by immunoelectron microscopy, despite lack of conventional secretory or cell‐surface anchorage signals within the primary sequence. Full‐length recombinant PavA polypeptide bound to immobilized human fibronectin in preference to fluid‐phase fibronectin, in a heparin‐sensitive interaction, and blocked binding of wild‐type pneumococcal cells to fibronectin. However, a C‐terminally truncated PavA′ polypeptide (362 aa residues) failed to bind fibronectin or block pneumococcal cell adhesion. Expression of pavA in Enterococcus faecalis JH2–2 conferred > sixfold increased cell adhesion levels to fibronectin over control JH2–2 cells. Isogenic mutants of S. pneumoniae, either abrogated in PavA expression or producing a 42 kDa C‐terminally truncated protein, showed up to 50% reduced binding to immobilized fibronectin. Inactivation of pavA had no effects on growth rate, cell morphology, cell‐surface physico‐chemical properties, production of pneumolysin, autolysin, or surface proteins PspA and PsaA. Isogenic pavA mutants of encapsulated S. pneumoniae D39 were approximately 104‐fold attenuated in virulence in the mouse sepsis model. These results provide evidence that PavA fibronectin‐binding protein plays a direct role in the pathogenesis of pneumococcal infections.

Streptococcus gordonii modulates Candida albicans biofilm formation through intergeneric communication.

ABSTRACT The fungus Candida albicans colonizes human oral cavity surfaces in conjunction with a complex microflora. C. albicans SC5314 formed biofilms on saliva-coated surfaces that in early stages of development consisted of ∼30% hyphal forms. In mixed biofilms with the oral bacterium Streptococcus gordonii DL1, hyphal development by C. albicans was enhanced so that biofilms consisted of ∼60% hyphal forms. Cell-cell contact between S. gordonii and C. albicans involved Streptococcus cell wall-anchored proteins SspA and SspB (antigen I/II family polypeptides). Repression of C. albicans hyphal filament and biofilm production by the quorum-sensing molecule farnesol was relieved by S. gordonii. The ability of a luxS mutant of S. gordonii deficient in production of autoinducer 2 to induce C. albicans hyphal formation was reduced, and this mutant suppressed farnesol inhibition of hyphal formation less effectively. Coincubation of the two microbial species led to activation of C. albicans mitogen-activated protein kinase Cek1p, inhibition of Mkc1p activation by H2O2, and enhanced activation of Hog1p by farnesol, which were direct effects of streptococci on morphogenetic signaling. These results suggest that interactions between C. albicans and S. gordonii involve physical (adherence) and chemical (diffusible) signals that influence the development of biofilm communities. Thus, bacteria may play a significant role in modulating Candida carriage and infection processes in the oral cavity.

PavA of Streptococcus pneumoniae Modulates Adherence, Invasion, and Meningeal Inflammation

ABSTRACT Pneumococcal adherence and virulence factor A (PavA) is displayed to the cell outer surface of Streptococcus pneumoniae and mediates pneumococcal binding to immobilized fibronectin. PavA, which lacks a typical gram-positive signal sequence and cell surface anchorage motif, is essential for pneumococcal virulence in a mouse infection model of septicemia. In this report the impact of PavA on pneumococcal adhesion to and invasion of eukaryotic cells and on experimental pneumococcal meningitis was investigated. In the experimental mouse meningitis model, the virulence of the pavA knockout mutant of S. pneumoniae D39, which did not show alterations of subcellular structures as indicated by electron microscopic studies, was strongly decreased. Pneumococcal strains deficient in PavA showed substantially reduced adherence to and internalization of epithelial cell lines A549 and HEp-2. Similar results were obtained with human brain-derived microvascular endothelial cells and human umbilical vein-derived endothelial cells. Attachment and internalization of pneumococci were not significantly affected by preincubation or cocultivations of pneumococci with anti-PavA antisera. Pneumococcal adherence was also not significantly affected by the addition of PavA protein. Complementation of the pavA knockout strain with exogenously added PavA polypeptide did not restore adherence of the mutant. These data suggest that PavA affects pneumococcal colonization by modulating expression or function of important virulence determinants of S. pneumoniae.

Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands.

The antigen I/II (AgI/II) family polypeptides, ranging from 1310 to 1653 amino acid (aa) residues, are cell wall anchored adhesins expressed by most indigenous species of oral streptococci. The polypeptides interact with a wide range of host molecules, in particular salivary agglutinin glycoprotein (SAG or gp340), and with ligands on other oral bacteria. To determine the receptor recognition properties of six different AgI/II family polypeptides from strains of Streptococcus gordonii, Streptococcus intermedius and Streptococcus mutans, the genes were cloned and expressed on the surface of the surrogate host Lactococcus lactis. The S. gordonii SspA and SspB polypeptides mediated higher binding levels of L. lactis cells to surface immobilized gp340 than did S. intermedius Pas protein, or S. mutans SpaP or PAc proteins. However, the AgI/II proteins were all similar in their abilities to mediate aggregation of lactococci by fluid phase gp340. The SpaPI polypeptide from S. mutans Ingbritt, which was C‐terminally truncated by approximately 400 aa residues, did not bind gp340. Lactococci expressing AgI/II proteins, including SpaPI, were aggregated by a synthetic 16 aa residue peptide SRCRP2 derived from the aa repeat block sequences within gp340. In coaggregation assays, SspB from S. gordonii was unique in mediating coaggregation with only group A and group E strains of Actinomyces naeslundii. All the other AgI/II polypeptides mediated coaggregation with group C and group D strains of A. naeslundii. Analysis of chimeric protein constructs revealed that coaggregation specificity was determined by sequences within the N‐terminal half of AgI/II protein. A synthetic peptide (20 aa residues), which defines a putative adhesion epitope within the C‐terminal region of polypeptide, inhibited AgI/II‐mediated aggregation by gp340 but did not affect coaggregation with A. naeslundii. These results suggest that different mechanisms operate in interactions of AgI/II family polypeptides with native gp340, gp340 SRCR domain peptide, and A. naeslundii. Specificity of these interactions appears to be determined by discontinuous but interacting regions of the polypeptides, thus providing flexibility in receptor recognition for streptococcal colonization of the human host.

Interaction of Candida albicans Cell Wall Als3 Protein with Streptococcus gordonii SspB Adhesin Promotes Development of Mixed-Species Communities

ABSTRACT Candida albicans colonizes human mucosa and prosthetic surfaces associated with artificial joints, catheters, and dentures. In the oral cavity, C. albicans coexists with numerous bacterial species, and evidence suggests that bacteria may modulate fungal growth and biofilm formation. Streptococcus gordonii is found on most oral cavity surfaces and interacts with C. albicans to promote hyphal and biofilm formation. In this study, we investigated the role of the hyphal-wall protein Als3p in interactions of C. albicans with S. gordonii. Utilizing an ALS3 deletion mutant strain, it was shown that cells were not affected in initial adherence to the salivary pellicle or in hyphal formation in the planktonic phase. However, the Als3− mutant was unable to form biofilms on the salivary pellicle or deposited S. gordonii DL1 wild-type cells, and after initial adherence, als3Δ/als3Δ (ΔALS3) cells became detached concomitant with hyphal formation. In coaggregation assays, S. gordonii cells attached to, and accumulated around, hyphae formed by C. albicans wild-type cells. However, streptococci failed to attach to hyphae produced by the ΔALS3 mutant. Saccharomyces cerevisiae S150-2B cells expressing Als3p, but not control cells, supported binding of S. gordonii DL1. However, S. gordonii Δ(sspA sspB) cells deficient in production of the surface protein adhesins SspA and SspB showed >50% reduced levels of binding to S. cerevisiae expressing Als3p. Lactococcus lactis cells expressing SspB bound avidly to S. cerevisiae expressing Als3p, but not to S150-2B wild-type cells. These results show that recognition of C. albicans by S. gordonii involves Als3 protein-SspB protein interaction, defining a novel mechanism in fungal-bacterial communication.