Tryptic Shaving of Staphylococcus aureus Unveils Immunodominant Epitopes on the Bacterial Cell Surface.

The opportunistic pathogen Staphylococcus aureus has become a major threat for human health and well-being by developing resistance to antibiotics and by fast evolution into new lineages that rapidly spread within the healthy human population. This calls for development of active or passive immunization strategies to prevent or treat acute phase infections. Since no such anti-staphylococcal immunization approaches are available for clinical implementation, the present studies were aimed at identifying new leads for their development. For this purpose, we profiled the cell-surface-exposed staphylococcal proteome under infection-mimicking conditions by combining two approaches for "bacterial shaving" with immobilized or soluble trypsin and subsequent mass spectrometry analysis of liberated peptides. In parallel, non-covalently cell-wall-bound proteins extracted with potassium thiocyanate and the exoproteome fraction were analyzed by gel-free proteomics. All data are available through ProteomeXchange accession PXD000156. To pinpoint immunodominant bacterial-surface-exposed epitopes, we screened selected cell-wall-attached proteins of S. aureus for binding of immunoglobulin G from patients who have been challenged by different types of S. aureus due to chronic wound colonization. The combined results of these analyses highlight particular cell-surface-exposed S. aureus proteins with highly immunogenic exposed epitopes as potential targets for development of protective anti-staphylococcal immunization strategies.

Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions.

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria.

A human monoclonal antibody targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus aureus bacteremia.

Due to substantial therapy failure and the emergence of antibiotic-resistant Staphylococcus aureus strains, alternatives for antibiotic treatment of S. aureus infections are urgently needed. Passive immunization using S. aureus-specific monoclonal antibodies (mAb) could be such an alternative to prevent and treat severe S. aureus infections. The invariantly expressed immunodominant staphylococcal antigen A (IsaA) is a promising target for passive immunization. Here we report the development of the human anti-IsaA IgG1 mAb 1D9, which was shown to bind to all 26 S. aureus isolates tested. These included both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively). Immune complexes consisting of IsaA and 1D9 stimulated human as well as murine neutrophils to generate an oxidative burst. In a murine bacteremia model, the prophylactic treatment with a single dose of 5 mg/kg 1D9 improved the survival of mice challenged with S. aureus isolate P (MSSA) significantly, while therapeutic treatment with the same dose did not influence animal survival. Neither prophylactic nor therapeutic treatment with 5 mg/kg 1D9 resulted in improved survival of mice with S. aureus USA300 (MRSA) bacteremia. Importantly, our studies show that healthy S. aureus carriers elicit an immune response which is sufficient to generate protective mAbs against invariant staphylococcal surface antigens. Human mAb 1D9, possibly conjugated to for example another antibody, antibiotics, cytokines or chemokines, may be valuable to fight S. aureus infections in patients.

Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains.

The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.

Host-pathogen interactions in epidermolysis bullosa patients colonized with Staphylococcus aureus.

Patients with the genetic blistering disease epidermolysis bullosa (EB) often have chronic wounds that can become colonized by different bacteria, especially the opportunistic pathogen Staphylococcus aureus. We therefore determined the S. aureus colonization rates in EB patients from the Netherlands by collecting swabs from their anterior nares, throats and wounds. Within a period of ∼2 years, more than 90% of the sampled chronic wounds of EB patients were found to be colonized by S. aureus. Molecular typing revealed that EB patients were not colonized by a single S. aureus type. Rather the S. aureus population structure in the sampled EB patients mirrored the local S. aureus population structure within the Netherlands. Furthermore, multiple types of S. aureus were found in close proximity to each other within individual chronic wounds, indicating that these S. aureus types are not mutually exclusive. Over time, strong fluctuations in the S. aureus types sampled from individual EB patients were observed. This high exposure to different S. aureus types is apparently reflected by high plasma levels of antistaphylococcal IgG's, especially in patients carrying multiple S. aureus types. It remains to be determined to what extent this strong immune response protects EB patients against serious staphylococcal infections. Lastly, further research is needed to define the impact of staphylococcal colonization of chronic wounds on the development, exacerbation and healing of such wounds in patients with EB.

Topography of distinct Staphylococcus aureus types in chronic wounds of patients with epidermolysis bullosa.

The opportunistic pathogen Staphylococcus aureus is known to interfere with wound healing and represents a significant risk factor for wound infections and invasive disease. It is generally assumed that one individual is predominantly colonized by one S. aureus type. Nevertheless, patients with the genetic blistering disease epidermolysis bullosa (EB) often carry multiple S. aureus types. We therefore investigated whether different S. aureus types are present in individual wounds of EB patients and, if so, how they are spatially distributed. The staphylococcal topography in chronic wounds was mapped by replica-plating of used bandages and subsequent typing of S. aureus isolates. Individual chronic wounds of five patients contained up to six different S. aureus types. Unexpectedly, distinct S. aureus types formed micro-colonies that were located in close proximity and sometimes even overlapped. While some adjacent S. aureus isolates were closely related, others belonged to distinct molecular complexes. We conclude that the general assumption that one individual is predominantly colonized by one type of S. aureus does not apply to chronic wounds of EB patients. We consider this observation important, not only for EB patients, but also for other patients with chronic wounds in view of the potential risk for severe staphylococcal infections.

Requirement of signal peptidase ComC and thiol-disulfide oxidoreductase DsbA for optimal cell surface display of pseudopilin ComGC in Staphylococcus aureus.

Staphylococcus aureus is an important Gram-positive bacterial pathogen producing many secreted and cell surface-localized virulence factors. Here we report that the staphylococcal thiol-disulfide oxidoreductase DsbA is essential for stable biogenesis of the ComGC pseudopilin. The signal peptidase ComC is indispensable for ComGC maturation and optimal cell surface exposure.

High genetic diversity of Staphylococcus aureus strains colonizing patients with epidermolysis bullosa.

Patients with the blistering disease, epidermolysis bullosa (EB), frequently suffer from chronic wounds that become colonized by pathogenic bacteria, such as Staphylococcus aureus. To determine S. aureus colonization rates in patients with EB, swabs were collected from the anterior nares, throats and wounds of 52 Dutch patients with EB. Swabs were also collected from nares and throats of 13 healthcare workers who occasionally meet the sampled patients with EB. All EB patients with chronic wounds and 75% of the patients without chronic wounds were colonized with S. aureus. In contrast, 39% of the sampled healthcare workers were colonized with S. aureus. Typing revealed a high degree of genetic diversity of 184 collected S. aureus isolates. Autoinoculation of S. aureus in individual patients with EB was shown to occur frequently, whereas transmission of S. aureus between patients with EB is apparently rare. There was no evidence for S. aureus transmission between patients with EB and healthcare workers.

Surface shaving as a versatile tool to profile global interactions between human serum proteins and the Staphylococcus aureus cell surface.

The human commensal bacterium Staphylococcus aureus is renowned as a causative agent of severe invasive diseases. Upon entering the bloodstream, S. aureus can infect almost every tissue and organ system in the human body. To withstand insults from the immune system upon invasion, several immune-evasive mechanisms have evolved in S. aureus, such as complement inhibition by secreted proteins and IgG-binding by surface-exposed protein A. While it is generally accepted that S. aureus cells bind a range of host factors for various purposes, no global analyses to profile staphylococcal host factor binding have so far been performed. Therefore, we explored the possibility to profile the binding of human serum proteins to S. aureus cells by "surface shaving" with trypsin and subsequent MS analysis of liberated peptides. This resulted in the identification of several components of the complement system, the platelet factor 4 and the isoform 1 of the inter-α-trypsin inhibitor heavy chain H4 on the staphylococcal cell surface. We conclude that surface shaving is a versatile tool to profile global interactions between human serum proteins and the S. aureus cell surface.

Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in Staphylococcus aureus.

The gram-positive pathogen Staphylococcus aureus secretes various proteins into its extracellular milieu. Bioinformatics analyses have indicated that most of these proteins are directed to the canonical Sec pathway, which consists of the translocation motor SecA and a membrane-embedded channel composed of the SecY, SecE, and SecG proteins. In addition, S. aureus contains an accessory Sec2 pathway involving the SecA2 and SecY2 proteins. Here, we have addressed the roles of the nonessential channel components SecG and SecY2 in the biogenesis of the extracellular proteome of S. aureus. The results show that SecG is of major importance for protein secretion by S. aureus. Specifically, the extracellular accumulation of nine abundant exoproteins and seven cell wall-bound proteins was significantly affected in an secG mutant. No secretion defects were detected for strains with a secY2 single mutation. However, deletion of secY2 exacerbated the secretion defects of secG mutants, affecting the extracellular accumulation of one additional exoprotein and one cell wall protein. Furthermore, an secG secY2 double mutant displayed a synthetic growth defect. This might relate to a slightly elevated expression of sraP, encoding the only known substrate for the Sec2 pathway, in cells lacking SecG. Additionally, the results suggest that SecY2 can interact with the Sec1 channel, which would be consistent with the presence of a single set of secE and secG genes in S. aureus.

Opposite effects of Mn2+ and Zn2+ on PsaR-mediated expression of the virulence genes pcpA, prtA, and psaBCA of Streptococcus pneumoniae.

Homeostasis of Zn(2+) and Mn(2+) is important for the physiology and virulence of the human pathogen Streptococcus pneumoniae. Here, transcriptome analysis was used to determine the response of S. pneumoniae D39 to a high concentration of Zn(2+). Interestingly, virulence genes encoding the choline binding protein PcpA, the extracellular serine protease PrtA, and the Mn(2+) uptake system PsaBC(A) were strongly upregulated in the presence of Zn(2+). Using random mutagenesis, a previously described Mn(2+)-responsive transcriptional repressor, PsaR, was found to mediate the observed Zn(2+)-dependent derepression. In addition, PsaR is also responsible for the Mn(2+)-dependent repression of these genes. Subsequently, we investigated how these opposite effects are mediated by the same regulator. In vitro binding of purified PsaR to the prtA, pcpA, and psaB promoters was stimulated by Mn(2+), whereas Zn(2+) destroyed the interaction of PsaR with its target promoters. Mutational analysis of the pcpA promoter demonstrated the presence of a PsaR operator that mediates the transcriptional effects. In conclusion, PsaR is responsible for the counteracting effects of Mn(2+) and Zn(2+) on the expression of several virulence genes in S. pneumoniae, suggesting that the ratio of these metal ions exerts an important influence on pneumococcal pathogenesis.

The novel transcriptional regulator SczA mediates protection against Zn2+ stress by activation of the Zn2+-resistance gene czcD in Streptococcus pneumoniae.

Maintenance of the intracellular homeostasis of metal ions is important for the virulence of many bacterial pathogens. Here, we demonstrate that the czcD gene of the human pathogen Streptococcus pneumoniae is involved in resistance against Zn2+, and that its transcription is induced by the transition-metal ions Zn2+, Co2+ and Ni2+. Upstream of czcD a gene was identified, encoding a novel TetR family regulator, SczA, that is responsible for the metal ion-dependent activation of czcD expression. Transcriptome analyses revealed that in a sczA mutant expression of czcD, a gene encoding a MerR-family transcriptional regulator and a gene encoding a zinc-containing alcohol dehydrogenase (adhB) were downregulated. Activation of the czcD promoter by SczA is shown to proceed by Zn2+-dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. This is the first example of a metalloregulatory protein belonging to the TetR family that has been described. The presence in S. pneumoniae of the Zn2+-resistance system characterized in this study might reflect the need for adjustment to a fluctuating Zn2+ pool encountered by this pathogen during infection of the human body.