Staphylococcus aureus TarP: A Brick in the Wall or Rosetta Stone?

Abstract

Staphylococcus aureus infection elicits antibodies against wall teichoic acid (WTA). Several glycosyltransferases modify WTA to generate anomeric heterogeneity. In recent work, Gerlach et al. (2018) show that modification by prophage-encoded TarP diminishes WTA immunogenicity, allowing staphylococci to evade host adaptive immune responses, and propose to exploit these insights for vaccines.

Peptidoglycan is the rigid extra-cytoskeletal armor that protects bacteria from lysis. It is often referred to as cell wall or murein and serves as a scaffold for the assembly of proteins and carbohydrates in the bacterial envelope. Peptidoglycan of Staphylococcus aureus and other gram-positive bacteria is modified with wall teichoic acid (WTA), an anionic glycopolymer that, in most S. aureus strains, is comprised of 40–60 ribitol phosphate (Rbo-P) subunits (Winstel et al., 2014). The hydroxyl-groups of Rbo-P WTA are modified by D-alanine (D-Ala) and N-acetylglucosamine (GlcNAc) residues. This provides resistance to host antimicrobial peptides (D-Ala) or confers susceptibility to bacteriophage infection and acquisition of S. aureus pathogenicity islands (SaPIs) carrying genes that contribute to pathogenesis in human and animal hosts (Winstel et al., 2014). Two types of WTA glycosylation had been described thus far: O-linked α- and β-GlcNAc at the C4 position of Rbo-P, which is catalyzed by TarM and TarS, respectively (Winstel et al., 2014). Gerlach and colleagues now report the discovery of a third enzyme, TarP, generating O-linked β-GlcNAc at the C3 position of Rbo-P WTA (Gerlach et al., 2018). The tarP gene is carried on bacteriophages lysogenizing some hospital-acquired (HAMRSA) or lifestock-associated methicillin-resistant S. aureus (LA-MRSA) isolates. tarP expression impedes TarS-catalyzed β-GlcNAc modification at C4 of Rbo-P, enhancing infection of S. aureus with podophages while simultaneously blocking infection with sinophages. The tarP gene was found on three prophages of HAMRSA CC5 isolates, and one of these (фtarP-Sa3int) also carries genes involved in bacterial evasion from human immune defenses, including scn (staphylococcal complement inhibitor), chp (chemotaxis inhibitory protein), sak (staphylokinase), and sep (enterotoxin P—a T cell superantigen). This prompted the authors to investigate whether the bacteriophage/SaPI dissemination determinant tarP may also contribute to S. aureus evasion from host immune responses.

S. aureus persistently colonizes the nasopharynx and gastrointestinal tract of one-third of the human population. The remainder of the population represents intermediate carriers of the pathogen. Colonization is the key risk factor for community- and hospital-acquired S. aureus infections (Wertheim et al., 2005). In the community, S. aureus causes predominantly skin and soft tissue infections (SSTIs), and also pneumonia, osteomyelitis, septic arthritis, and bloodstream infections (Tong et al., 2015). In hospitals and other healthcare settings, S. aureus is most frequently associated with surgical site infections (SSIs), infections of implanted medical devices, respiratory tract infections in individuals requiring mechanical ventilation, and bloodstream infections in end-stage renal disease patients requiring hemodialysis (Tong et al., 2015).S. aureus colonization is associated with the development of serum antibody responses (predominantly IgG4) against some of the secreted antigens of S. aureus. These antibody responses are not known to impact S. aureus colonization, nor are they protective against S. aureus invasive diseases (Wertheim et al., 2005). Annual attack rates for the most abundant S. aureus diseases (SSTI and SSI) affect approximately 1% of the target population and recurrent disease (mostly relapses with the index strain following antibiotic and/or surgical intervention) is frequent (Tong et al., 2015). Analysis of heritable diseases with increased incidence of S. aureus infection points to the key role of human neutrophils as the first line of defense (Spaan et al., 2013). While S. aureus invasion of host tissues triggers neutrophil chemotaxis, complement activation, and opsonization, as well as phagocytosis, staphylococci secrete a plethora of factors that interfere with each of the neutrophil steps intended to clear the bacteria via phagocytic killing (Spaan et al., 2013). The individual contributions of these immune evasion factors can be assessed by inoculating wild-type and mutant S. aureus strains into freshly drawn human blood. Whereas wild-type clinical isolates largely resist opsonophagocytic killing (OPK) by human neutrophils, mutants exhibit varying degrees of susceptibility that rely on mutational defects in specific virulence genes. Although many S. aureus virulence determinants evolved to interfere with human components of the innate and adaptive immune system, others act more broadly, interfering with defenses of humans and of specific animal species (Spaan et al., 2013). For the latter, gene contributions toward staphylococcal virulence can be assessed by quantifying disease processes in animals that have been inoculated with wild-type and mutant S. aureus strains.

Early microbiologists developed the precipitin test, which enabled the discovery and quantification of human and animal antibody responses against bacterial pathogens (Dochez and Avery, 1917). Analyzing human sera from convalescents of S. aureus disease and purified components of the staphylococcal cell wall, Baddiley and Strominger demonstrated that the bulk of anti-S. aureus antibodies bound WTA, specifically the GlcNAc modification of Rbo-P (Nathenson and Strominger, 1962). Similar results were observed when immunizing rabbits or donkeys with heat-killed S. aureus. In agreement with these early studies, analysis of cloned antibodies from plasmablasts, B cell antibody factories circulating in the blood of patients with S. aureus infection, revealed an abundance of IgG that binds to GlcNAc moieties of RboP (Lehar et al., 2015). These purified monoclonal antibodies bind to the microbial surface and promote opsonophagocytosis. However, they fail to trigger killing of S. aureus, which can be attributed to bacterial resistance mechanisms against neutro-phil antimicrobial enzymes, reactive oxygen species, and nitrosative (Lehar et al., 2015). Further, passive administration of monoclonal antibodies specific for α- or β-glycosylated WTA antibodies does not protect mice against MRSA bloodstream challenge (Lehar et al., 2015). Immune globulin intravenous (IGIV, pooled immunoglobulin preparations from humans), which contains 0.3 mg/mL anti-S. aureus antibodies, two-thirds of which bind to glycosylated WTA, cannot protect animals against MRSA challenge either. (Lehar et al., 2015). Earlier work examined the role of IVIG in preventing nosocomialS. aureus sepsis in very-low-birth-weight neonates (501–1,500 g of birth weight). However, the study failed to demonstrate protective efficacy (Fanaroff et al., 1994).

Binding of human antibodies against S. aureus is diminished for mutants lacking WTA glycosylation, and this defect can be restored by plasmid-borne expression of tarS (Kurokawa et al., 2013). Gerlach and colleagues show that antibody binding is only marginally increased when expressing tarP in the ΔtarS ΔtarP mutant S. aureus. Further, TarP-mediated glycosylation of WTA (β-GlcNAc at Rbo-P C3) is dominant over that of TarS (β-GlcNAc at Rbo-P C4). The authors also observed increased levels of antibody binding to ΔtarP as compared to wild-type N315. Immunization of mice with purified, aluminum-hydroxide adjuvanted WTA from wild-type S. aureus N315 (tarS⁺, tarP⁺) or its ΔtarS and ΔtarP variants did not protect mice against bacterial replication in renal tissue following intravenous challenge with N315. From these data, the authors propose that TarP modification of WTA with β-GlcNAc at Rbo-P C3 may subvert antibody responses against S. aureus WTA that would otherwise be modified with β-GlcNAc at Rbo-P C4 (TarS). The authors speculate further that specific inhibitors of TarP may overcome the presumed immune evasive effects of its expression in S. aureus tarS⁺ strains and that this could be useful in conjunction vaccines that elicit WTA-specific antibodies.

The TarP-derived mechanism of WTA glycosylation (β-GlcNAc at Rbo-P C3 and interference with TarS-catalyzed glycosylation at C4) certainly represents an interesting strategy of staphylococci to modify dissemination of specific bacteriophages and SaPIs among staphylococci. In agreement with this conjecture, tarS, tarM, and tarP are found not only in the genome sequences of S. aureus but also in staphylococcal species not known to cause human or animal disease. tarP is located in close proximity to the integrase gene of bacteriophages but is not associated with the immune evasion cluster (scn, chp, sak, sep) of фtarP-Sa3int, the only example for an SaPI carrying both tarP and known immune evasion genes. Occurrence of tarP carrying bacteriophages and SaPIs in S. aureus strains causing human disease is rare and interference would appear to be limited to antibodies recognizing the TarS-catalyzed β-GlcNAc modification at Rbo-P C4. These antibodies are, however, not known to be associated with immunity against human S. aureus diseases. Thus, additional exploratory and experimental work is necessary to substantiate the hypothesis of Gerlach and colleagues that TarP represents a new type of Rosetta Stone to unravel the conundrum of S. aureus immune evasion and discover vaccines with protective efficacy. Nevertheless, TarP does add a new type of brick to the cell wall of staphylococci, thereby modifying the horizontal gene transfer with bacteriophages and SaPIs among staphylococci.