金黄色葡萄球菌生物膜形成机制研究进展

金黄色葡萄球菌是医院和社区获得性感染最常见的病原菌之一,而且可形成生物膜,从而导致生物膜相关疾病的产生。患者一旦发生金黄色葡萄球菌生物膜感染,难以彻底治愈。深入研究金黄色葡萄球菌生物膜形成的分子机制和调控网络,对寻找有效的防治、治疗药物和手段具有重要意义。我们就金黄色葡萄球菌生物膜形成过程和调控机制的研究近况做一综述。     

SaeRS-Dependent Inhibition of Biofilm Formation in Staphylococcus aureus Newman

The SaeRS two-component regulatory system of Staphylococcus aureus is known to affect the expression of many genes. The SaeS protein is the histidine kinase responsible for phosphorylation of the response regulator SaeR. In S. aureus Newman, the sae system is constitutively expressed due to a point mutation in saeS, relative to other S. aureus strains, which results in substitution of proline for leucine at amino acid 18. Strain Newman is unable to form a robust biofilm and we report here that the biofilm-deficient phenotype is due to the saeS^P allele. Replacement of the Newman saeS^P with saeS^L, or deletion of saeRS, resulted in a biofilm-proficient phenotype. Newman culture supernatants were observed to inhibit biofilm formation by other S. aureus strains, but did not affect biofilm formation by S. epidermidis. Culture supernatants of Newman saeS^L or Newman ΔsaeRS had no significant effect on biofilm formation. The inhibitory factor was inactivated by incubation with proteinase K, but survived heating, indicating that the inhibitory protein is heat-stable. The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms. Replacement of saeS^L with saeS^P in the biofilm-proficient S. aureus USA300 FPR3757 resulted in the loss of biofilm formation. Culture supernatants of USA300 FPR3757 saeS^P, did not inhibit biofilm formation by other staphylococci, suggesting that the inhibitory factor is produced but not secreted in the mutant strain. A number of biochemical methods were utilized to isolate the inhibitory protein. Although a number of candidate proteins were identified, none were found to be the actual inhibitor. In an effort to reduce the number of potential inhibitory genes, RNA-Seq analyses were done with wild-type strain Newman and the saeS^L and ΔsaeRS mutants. RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.     

Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation

Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α₂-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.     

d-Amino Acids Do Not Inhibit Biofilm Formation in Staphylococcus aureus

Bacteria can either exist in the planktonic (free floating) state or in the biofilm (encased within an organic framework) state. Bacteria biofilms cause industrial concerns and medical complications and there has been a great deal of interest in the discovery of small molecule agents that can inhibit the formation of biofilms or disperse existing structures. Herein we show that, contrary to previously published reports, d-amino acids do not inhibit biofilm formation of Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), and Staphylococcus epidermis (S. epidermis) at millimolar concentrations. We evaluated a diverse set of natural and unnatural d-amino acids and observed no activity from these compounds in inhibiting biofilm formation.     

Role of Surface Protein SasG in Biofilm Formation by Staphylococcus aureus

The SasG surface protein of Staphylococcus aureus has been shown to promote the formation of biofilm. SasG comprises an N-terminal A domain and repeated B domains. Here we demonstrate that SasG is involved in the accumulation phase of biofilm, a process that requires a physiological concentration of Zn²⁺. The B domains, but not the A domain, are required. Purified recombinant B domain protein can form dimers in vitro in a Zn²⁺-dependent fashion. Furthermore, the protein can bind to cells that have B domains anchored to their surface and block biofilm formation. The full-length SasG protein exposed on the cell surface is processed within the B domains to a limited degree, resulting in cleaved proteins of various lengths being released into the supernatant. Some of the released molecules associate with the surface-exposed B domains that remain attached to the cell. Studies using inhibitors and mutants failed to identify any protease that could cause the observed cleavage within the B domains. Extensively purified recombinant B domain protein is very labile, and we propose that cleavage occurs spontaneously at labile peptide bonds and that this is necessary for biofilm formation.Staphylococcus aureus is a commensal bacterium that is carried persistently in the anterior nares of about 20% of the human population. The organism can cause superficial skin infections, such as abscesses and impetigo, and more dangerous and potentially life-threatening invasive infections, such as endocarditis, osteomyelitis, and septic arthritis (26). Staphylococcus epidermidis and S. aureus are the major causes of infections associated with indwelling medical devices, such as central venous catheters, cardiovascular devices, and artificial joints (34, 54). The ability to form a biofilm is crucial to the microbes'' success in device-related infections. Bacteria in the biofilm matrix are in a semidormant state, are difficult to inhibit with antibiotics, and are impervious to host neutrophils and macrophages (36, 43, 44, 51). Until recently biofilm formation by staphylococci was attributed to the ability to synthesize an extracellular polysaccharide called polysaccharide intercellular adhesin (PIA), which is composed of partially deacetylated poly-N-acetylglucosamine (15, 28, 50). Attachment of bacteria to biomedical devices is mediated by adhesion to the naked plastic or metal surface by a surface component such as the major autolysin Atl (2, 14). Alternatively, adhesion to surfaces that have been conditioned by fibronectin and fibrinogen from host plasma is mediated by surface proteins such as clumping factor A (ClfA) and fibronectin binding proteins (FnBPA/B) of S. aureus or SdrG/Fbe of S. epidermidis (17, 46, 47).Several surface proteins of staphylococci can also promote the accumulation phase of biofilm: (i) the biofilm-associated protein Bap, which is only expressed by bovine strains of S. aureus (8); (ii) the SasC surface protein of S. aureus (41); (iii) fibronectin binding proteins FnBPA and FnBPB, which are particularly associated with biofilm formation by some types of methicillin-resistant S. aureus (MRSA) (35, 48); (iv) the multifactorial virulence factor protein A, which promotes cell accumulation when expressed at high levels, for example,in mutants defective in the accessory gene regulator Agr (31); (v) the extracellular matrix binding protein (Embp) of S. epidermidis (4); (vi) the accumulation-associated protein (Aap) of S. epidermidis and the related protein SasG from S. aureus (7, 19, 40).Aap and SasG are typical LPXTG-anchored multidomain cell wall-associated proteins (see Fig. ​Fig.1A,1A, below). A signal sequence is removed from the N terminus during secretion across the cytoplasmic membrane. The C-terminal domains comprise a sorting signal (LPXTG) and hydrophobic membrane-spanning domain and positively charged residues that are required for covalent attachment of the proteins to cell wall peptidoglycan by sortase A. The N termini of the mature proteins (A domains) comprise related amino acid sequences that have been implicated in adhesion of bacteria to desquamated epithelial cells and could be involved in colonization of the nares and skin (7, 27, 39). The archetypal Aap protein of S. epidermidis RP62a has 12 repeats of almost identical sequences of 128 residues followed by a partial repeat of 68 residues (region B), while SasG from S. aureus strain 8325-4 and strain Newman has seven 128-residue repeats and one partial repeat. The B subunits of Aap and SasG are 64% identical.Open in a separate windowFIG. 1.(A) Schematic representation of SasG domain organization. The positions of the signal sequence (S), A domain, B region (B1 to -8), and the wall/membrane-spanning regions (W/M) are indicated. The LPKTG motif is recognized by the sortase A enzyme, which covalently anchors the protein to the cell wall peptidoglycan. (B) Whole-cell immunoblot validating expression of A domain and B regions of SasG variants. Serial dilutions of SH1000(pALC2073:sasG⁺) (row 1); SH1000(pALC2073sasG⁺ A⁺B⁻) (row 2); SH1000(pALC2073sasG⁺ A⁻B⁺) (row 3), and SH1000(pALC2073sasG⁺ A⁻B⁺) induced with tetracycline (90 ng/ml) (row 4) were applied to a nitrocellulose membrane and probed with anti-SasG A domain and anti-SasG B domain antibodies. (C) Biofilm formation by SH1000 constructs expressing SasG variants. Biofilm was allowed to form for 24 h at 37°C under static conditions in microtiter dishes. Biofilm was stained with crystal violet, and the absorbance was measured at 570 nm.The formation of biofilm by Aap in S. epidermidis is promoted by the removal of the A domain by cleavage by an as-yet-unidentified bacterial protease, an event that can also be precipitated by host proteases (40). The ability of the exposed Aap B domains of different bacterial cells to form homophilic interactions through a Zn²⁺-dependent zipper mechanism was proposed when it was shown that purified B domains formed dimers in vitro that were dependent on the presence of Zn²⁺ (6). Purified recombinant B domain protein, but not the A domain, inhibited biofilm formation, as did antibodies that specifically bound to the B domains (40). The Zn²⁺ chelator diethylenetriaminepentaacetic acid (DTPA) inhibited biofilm formation both by S. epidermidis RP62a (presumed to be due to Aap) and by community-associated MRSA (presumed to be due to SasG) (6).This study set out to investigate the molecular basis of biofilm accumulation promoted by the SasG protein of S. aureus. We demonstrate that processing of SasG occurs during growth and biofilm formation in a manner that is different from that reported for Aap, and we have investigated the mechanism.     

Novel Inhibitors of Staphylococcus aureus Virulence Gene Expression and Biofilm Formation

Staphylococcus aureus is a major human pathogen and one of the more prominent pathogens causing biofilm related infections in clinic. Antibiotic resistance in S. aureus such as methicillin resistance is approaching an epidemic level. Antibiotic resistance is widespread among major human pathogens and poses a serious problem for public health. Conventional antibiotics are either bacteriostatic or bacteriocidal, leading to strong selection for antibiotic resistant pathogens. An alternative approach of inhibiting pathogen virulence without inhibiting bacterial growth may minimize the selection pressure for resistance. In previous studies, we identified a chemical series of low molecular weight compounds capable of inhibiting group A streptococcus virulence following this alternative anti-microbial approach. In the current study, we demonstrated that two analogs of this class of novel anti-virulence compounds also inhibited virulence gene expression of S. aureus and exhibited an inhibitory effect on S. aureus biofilm formation. This class of anti-virulence compounds could be a starting point for development of novel anti-microbial agents against S. aureus.     

Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation

Staphylococcus aureus biofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role in S. aureus biofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating on S. aureus biofilm formation was tested with in vitro microplate biofilm assays and an in vivo mouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by various S. aureus strains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics. In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduce S. aureus biofilm formation both in vitro and in vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices.     

Effect of High Oxygen Concentration on Virulence of Staphylococcus aureus

Cultures of Staphylococcus aureus were agitated and treated with oxygen or air for a period of 29 days. At intervals of 1, 3, 9, 15, and 29 days, samples of washed cells were tested for coagulase activity and for abscess-producing ability. It was found that aeration with agitation initially increased abscess formation and that the increase was of the same magnitude for oxygen and air. Continued treatment beyond 1 day resulted in a progressive decrease in this activity which was more pronounced in the oxygenated cultures. Throughout the treatment, the bound coagulase activity remained constant. Thus, there appeared to be no quantitative relationship of coagulase titer with virulence as expressed by lesion formation.     

黄芩素抑制金黄色葡萄球菌生物被膜的形成

细菌生物被膜的形成是导致细菌耐药和引起持续性感染的主要原因之一。本文通过检测黄芩素对金黄色葡萄球菌26112菌株（Staphylococcus aureus 26112,SA26112）多糖细胞间黏附素（polysaccharide intercellular adhesion, PIA）的合成和胞外DNA（extracellular DNA,eDNA）释放量的影响,及其对icaA和cidA基因表达量的影响,探讨黄芩素对金黄色葡萄菌生物被膜形成的抑制作用及其机制。结果显示,黄芩素能抑制SA26112生物被膜的形成,其抑杀SA26112的最低抑菌浓度和最低杀菌浓度均为0.04 mg/mL。0.16 mg/mL黄芩素和256 μg/mL环丙沙星单独作用时,均不能杀死其成熟生物被膜内的SA26112细菌,而当二者联用时则可杀死成熟生物被膜内的细菌。黄芩素能显著抑制SA26112菌株PIA的合成、eDNA的释放量及icaA和cidA基因的相对表达量。其中,0.04 mg/mL黄芩素作用SA26112菌株24 h,与对照组相比,eDNA的释放量减少97%,icaA和cidA基因的相对表达量分别减少62%和41%。上述结果表明,黄芩素能抑制SA26112菌株生物被膜的形成,其作用机制可通过降低icaA和cidA的基因表达量,进而影响PIA的合成和eDNA的释放,来抑制金黄色葡萄球菌生物被膜的形成。     

Baicalein Inhibits Staphylococcus aureus Biofilm Formation and the Quorum Sensing System In Vitro

Biofilm formed by Staphylococcus aureus significantly enhances antibiotic resistance by inhibiting the penetration of antibiotics, resulting in an increasingly serious situation. This study aimed to assess whether baicalein can prevent Staphylococcus aureus biofilm formation and whether it may have synergistic bactericidal effects with antibiotics in vitro. To do this, we used a clinically isolated strain of Staphylococcus aureus 17546 (t037) for biofilm formation. Virulence factors were detected following treatment with baicalein, and the molecular mechanism of its antibiofilm activity was studied. Plate counting, crystal violet staining, and fluorescence microscopy revealed that 32 μg/mL and 64 μg/mL baicalein clearly inhibited 3- and 7-day biofilm formation in vitro. Moreover, colony forming unit count, confocal laser scanning microscopy, and scanning electron microscopy showed that vancomycin (VCM) and baicalein generally enhanced destruction of biofilms, while VCM alone did not. Western blotting and real-time quantitative polymerase chain reaction analyses (RTQ-PCR) confirmed that baicalein treatment reduced staphylococcal enterotoxin A (SEA) and α-hemolysin (hla) levels. Most strikingly, real-time qualitative polymerase chain reaction data demonstrated that 32 μg/mL and 64 μg/mL baicalein downregulated the quorum-sensing system regulators agrA, RNAIII, and sarA, and gene expression of ica, but 16 μg/mL baicalein had no effect. In summary, baicalein inhibited Staphylococcus aureus biofilm formation, destroyed biofilms, increased the permeability of vancomycin, reduced the production of staphylococcal enterotoxin A and α-hemolysin, and inhibited the quorum sensing system. These results support baicalein as a novel drug candidate and an effective treatment strategy for Staphylococcus aureus biofilm-associated infections.     

The Effect of Brominated Furanones on The Formation of Staphylococcus aureus Biofilm on PVC

To study the influence of brominated furanones on the formation of Staphylococcus aureus (SA) biofilm on PVC thus providing new avenues of research on the surface modification of materials and clinical treatment of biomaterial-centered infection. Three brominated furanones (furanone-1, furanone-2, and furanone-3) were coated on the surface of PVC material. Both the modified PVC materials and SA were co-cultivated together. To assess the thickness of bacterial biofilm and bacterium colony unit area on PVC materials, confocal laser scanning microscopy and scanning electron microscopy (SEM) were used to observe the surface structure of SA biofilm formation. All treatments were compared with the control group which was not coated with furanones. PVC materials coated with furanone-1 had an increase in bacterial biofilm as well as SA colony area when compared with control. However, there was no significant difference between treating with furanone-1 and furanone-3 (P > 0.05). The impact of different brominated furanones on SA biofilm formation on the surface of PVC materials is different, furanone-1 can promote the SA biofilm formation on the surface of PVC material.     

Antibody-Mediated Immobilization of Cryptococcus neoformans Promotes Biofilm Formation

Most microbes, including the fungal pathogen Cryptococcus neoformans, can grow as biofilms. Biofilms confer upon microbes a range of characteristics, including an ability to colonize materials such as shunts and catheters and increased resistance to antibiotics. Here, we provide evidence that coating surfaces with a monoclonal antibody to glucuronoxylomannan, the major component of the fungal capsular polysaccharide, immobilizes cryptococcal cells to a surface support and, subsequently, promotes biofilm formation. We used time-lapse microscopy to visualize the growth of cryptococcal biofilms, generating the first movies of fungal biofilm growth. We show that when fungal cells are immobilized using surface-attached specific antibody to the capsule, the initial stages of biofilm formation are significantly faster than those on surfaces with no antibody coating or surfaces coated with unspecific monoclonal antibody. Time-lapse microscopy revealed that biofilm growth was a dynamic process in which cells shuffled position during budding and was accompanied by emergence of planktonic variant cells that left the attached biofilm community. The planktonic variant cells exhibited mobility, presumably by Brownian motion. Our results indicate that microbial immobilization by antibody capture hastens biofilm formation and suggest that antibody coating of medical devices with immunoglobulins must exclude binding to common pathogenic microbes and the possibility that this effect could be exploited in industrial microbiology.Cryptococcus neoformans is a fungal pathogen that is ubiquitous in the environment and enters the body via the inhalation of airborne particles. The C. neoformans cell is surrounded by a layer of polysaccharide that consists predominantly of glucuronoxylomannan (GXM), which forms a protective capsule around the microbe. The capsule has been shown to be essential for virulence in murine models of infection (5-7) and, thus, is considered a key virulence factor. C. neoformans is the causative agent of cryptococcosis, a disease that primarily affects individuals with impaired immune systems, and is a significant problem in AIDS patients (21, 31). The most common manifestation of cryptococcosis is meningoencephalitis.Biofilms are communities of microbes that are attached to surfaces and held together by an extracellular matrix, often consisting predominantly of polysaccharides (8, 10). A great deal is known about bacterial biofilms (3, 9, 24, 30), but fungal biofilm formation is much less studied. Candida albicans is known to synthesize biofilms (11, 28, 29), as is C. neoformans. Biofilm-like structures consisting of innumerable cryptococcal cells encased in a polysaccharide matrix have been reported in human cases of cryptococcosis (32). Biofilm formation confers upon the microbe the capacity for drug resistance, and microbial cells in biofilms are less susceptible to host defense mechanisms (2, 4, 9, 12). In this regard, cells within C. neoformans biofilms are significantly less susceptible to caspofungin and amphotericin B than are planktonic cells (19). The cells within the biofilm are also resistant to the actions of fluconazole and voriconazole and various microbial oxidants and peptides (17, 19).Bacterial and fungal biofilms form readily on prosthetic materials, which poses a tremendous risk of chronic infection (10, 13, 15, 27). C. neoformans biofilms can form on a range of surfaces, including glass, polystyrene, and polyvinyl, and material devices, such as catheters (16). C. neoformans can form biofilms on the ventriculoatrial shunts used to decompress intracerebral pressure in patients with cryptococcal meningoencephalitis (32).The polysaccharide capsule of C. neoformans is essential for biofilm formation (18), and biofilm formation involves the shedding and accumulation of large amounts of GXM into the biofilm extracellular matrix (16). Previously, we reported that antibody to GXM in solution could inhibit biofilm formation through a process that presumably involves interference with polysaccharide shedding (18, 20). However, the effect of antibody-mediated immobilization of C. neoformans cells on cryptococcal biofilm formation has not been explored. In this paper, we report that the monoclonal antibody (MAb) 18B7, which is specific for the capsular polysaccharide GXM, can capture and immobilize C. neoformans to surfaces, a process that promotes biofilm formation. Interestingly, we identified planktonic variant C. neoformans cells that appeared to escape from the biofilm, but whose functions are not known. The results provide new insights on biofilm formation.     

Beta-Hemolysin Promotes Skin Colonization by Staphylococcus aureus

Colonization by Staphylococcus aureus is a characteristic feature of several inflammatory skin diseases and is often followed by epidermal damage and invasive infection. In this study, we investigated the mechanism of skin colonization by a virulent community-acquired methicillin-resistant S. aureus (CA-MRSA) strain, MW2, using a murine ear colonization model. MW2 does not produce a hemolytic toxin, beta-hemolysin (Hlb), due to integration of a prophage, ϕSa3mw, inside the toxin gene (hlb). However, we found that strain MW2 bacteria that had successfully colonized murine ears included derivatives that produced Hlb. Genome sequencing of the Hlb-producing colonies revealed that precise excision of prophage ϕSa3mw occurred, leading to reconstruction of the intact hlb gene in their chromosomes. To address the question of whether Hlb is involved in skin colonization, we constructed MW2-derivative strains with and without the Hlb gene and then subjected them to colonization tests. The colonization efficiency of the Hlb-producing mutant on murine ears was more than 50-fold greater than that of the mutant without hlb. Furthermore, we also showed that Hlb toxin had elevated cytotoxicity for human primary keratinocytes. Our results indicate that S. aureus Hlb plays an important role in skin colonization by damaging keratinocytes, in addition to its well-known hemolytic activity for erythrocytes.     

Combined Use of Bacteriophage K and a Novel Bacteriophage To Reduce Staphylococcus aureus Biofilm Formation

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms.