An embarrassment of sortases - a richness of substrates?

A range of surface proteins is anchored to the cell walls of Gram-positive pathogens such as Staphylococcus aureus by the transpeptidase sortase. Until now, sortase-like proteins and their substrates appeared to be limited mainly to such pathogens. However, by searching for sortase homologues among complete and incomplete genome sequences, we have found them to be present in almost all Gram-positives, a single Gram-negative bacterium and an archaean. There is usually more than one sortase-like protein encoded in each Gram-positive genome, and the genes encoding the sortase-like proteins are often clustered with genes encoding their likely substrates.     

Iron acquisition by Gram-positive bacterial pathogens

For the majority of bacterial pathogens, acquisition of iron from host proteins is a prerequisite for growth during infection. The mechanisms by which Gram-negative bacteria obtain iron from host proteins have been well described, but only recently has substantial progress been made in identifying these mechanisms for Gram-positive bacterial pathogens. This review provides an overview of the existing knowledge on the genetic basis of iron transport for important Gram-positive pathogens.     

Virulence mechanisms of Gram-positive plant pathogenic bacteria

Actinobacteria and Firmicutes comprise a group of highly divergent prokaryotes known as Gram-positive bacteria, which are ancestral to Gram-negative bacteria. Comparative genomics is revealing that, though plant virulence genes are frequently located on plasmids or in laterally acquired gene clusters, they are rarely shared with Gram-negative bacterial plant pathogens and among Gram-positive genera. Gram-positive bacterial pathogens utilize a variety of virulence strategies to invade their plant hosts, including the production of phytotoxins to allow intracellular and intercellular replication, production of cytokinins to generate gall tissues for invasion, secretion of proteins to induce cankers and the utilization and manipulation of sap-feeding insects for introduction into the phloem sieve cells. Functional analysis of novel virulence genes utilized by Actinobacteria and Firmicutes is revealing how these ancient prokaryotes manipulate plant, and sometimes insect, metabolic processes for their own benefit.     

1H, 13C and 15N resonance assignments of the major extracytoplasmic domain of the cell shape-determining protein MreC from Bacillus subtilis

MreB, MreC and MreD are essential cell shape-determining morphogenetic proteins in Gram-positive and in Gram-negative bacteria. While MreB, the bacterial homologue of the eukaryotic cytoskeletal protein actin, has been extensively studied, the roles of MreC and MreD are less well understood. They both are transmembrane proteins. MreC has a predicted single transmembrane domain and the C-terminal part outside the cell membrane. MreC probably functions as a link between the intracellular cytoskeleton and the cell wall synthesizing machinery which is located at the outer surface of the cell membrane. Also proteins involved in cell wall synthesis participate in cell morphogenesis. How these two processes are coordinated is, however, poorly understood. Bacillus subtilis (BS), a non-pathogenic Gram-positive bacterium, is widely used as a model for Gram-positive pathogens, e.g. Staphylococcus aureus (SA). Currently, the structures of MreC from BS and SA are not known. As part of our efforts to elucidate the structure–function relationships of the morphogenetic protein complexes in Gram-positive bacteria, we present the backbone and side chain resonance assignments of the extracytoplasmic domain of MreC from BS.     

Benfang Lei's research on heme acquisition in Gram-positive pathogens and bacterial pathogenesis

Benfang Lei's laboratory conducts research on pathogenesis of human pathogen Group A Streptococcus (GAS) and horse pathogen Streptococcus equi (S. equi). His current research focuses on heme acquisition in Gram-positive pathogens and molecular mechanism of GAS and S. equi pathogenesis. Heme is an important source of essential iron for bacterial pathogens. Benfang Lei and colleagues identified the first cell surface heme-binding protein in Gram-positive pathogens and the heme acquisition system in GAS, demonstrated direct heme transfer from one protein to another, demonstrated an experimental pathway of heme acquisition by the Staphylococcus aureus Isd system, elucidated the activated heme transfer mechanism, and obtained evidence for a chemical mechanism of direct axial ligand displacement during the Shp-to-HtsA heme transfer reaction. These findings have considerably contributed to the progress that has been made over recent years in understanding the heme acquisition process in Gram-positive pathogens. Pathogenesis of GAS is mediated by an abundance of extracellular proteins, and pathogenic role and functional mechanism are not known for many of these virulence factors. Lei laboratory identified a secreted protein of GAS as a CovRS-regulated virulence factor that is a protective antigen and is critical for GAS spreading in the skin and systemic dissemination. These studies may lead to development of novel strategies to prevent and treat GAS infections.     

Assembly of pili on the surface of Corynebacterium diphtheriae

Pili of Gram-negative pathogens are formed from pilin precursor molecules by non-covalent association within the outer membrane envelope. Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, however, an assembly pathway for pili has not yet been revealed. We show here that pili of Corynebacterium diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Assembled pili are released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope. All three pilin subunit proteins are synthesized as precursors carrying N-terminal signal peptides and C-terminal sorting signals. Some, but not all, of the six sortase genes encoded in the genome of C. diphtheriae are required for precursor processing, pilus assembly or cell wall envelope attachment. Pilus assembly is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits. This covalent tethering of adjacent pilin subunits appears to have evolved in many Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs.     

利用固定化金属亲和层析和质谱技术鉴定化脓性链球菌铜结合蛋白

化脓性链球菌是一种常见的高致病性革兰氏阳性菌,每年都在全球范围内引发大量的感染现象与病死案例。金属蛋白在微生物的代谢以及毒性构成中的作用已经逐渐得到重视。通过固定化金属离子亲合层析技术对化脓性链球菌中铜结合蛋白进行分离,配合质谱技术的使用,共鉴定出21个铜结合蛋白,这些蛋白分别在翻译、代谢、离子通道等各个生理进程中发挥相应作用。     

一种抗革兰氏阳性致病菌新型靶酶——分选酶

许多革兰氏阳性菌的表面蛋白是经过一种被称为分选酶的半胱氨酸转肽酶的作用而锚定到细胞壁上,由于表面蛋白在病原菌的致病性方面起关键作用,分选酶有可能成为降低革兰氏阳性菌致病性的药物靶点.目前,通过对金黄色葡萄球菌中分选酶A(SrtA)的研究,已初步阐明分选酶的作用机制及其活性位点,与此同时,一些SrtA抑制剂的初步鉴定为今后抑制剂更深层次的筛选提供了基础.     

The interaction of macrophage receptors with bacterial ligands

Innate immune receptors play a key role in the early recognition of invading bacterial pathogens and initiate the crucial innate immune response. The diverse macrophage receptors recognise Gram-positive and Gram-negative bacteria via conserved structures on the bacterial surface and facilitate phagocytosis and/or signalling, providing the trigger for the adaptive immune response. These receptors include scavenger receptors, C-type lectins, integrins, Toll-like receptors and siglecs. The bacterial ligands generally recognised by these receptors range from lipopolysaccharides on Gram-negative bacteria to peptidoglycan and lipoteichoic acid on Gram-positive bacteria. However, emerging evidence indicates that bacterial proteins are also important ligands; for example, surface proteins from Neisseria meningitidis have been shown to be ligands for class A scavenger receptors. In addition, a group of cytosolic receptors, the NBS-LRR proteins, have been implicated in recognition of bacterial breakdown products. It is becoming increasingly apparent that macrophage receptors can act in conjunction with one another to deliver an appropriate response.     

Synthesis and structure-activity relationship of 7-(substituted)-aminomethyl-4-quinolone-3-carboxylic acid derivatives

Gram-positive organisms have re-emerged as the major hospital pathogens, which make the unmet medical needs for antibacterial therapy even worse. In searching for potent agents against Gram-positive pathogens, novel 7-(substituted)-aminomethyl-quinolone-3-carboxylic acids were designed, synthesized, and evaluated for their antibacterial activities in vitro. Many 7-monoarylaminomethyl derivatives exhibited high potency against Gram-positive organisms compared to reference agents: vancomycin and pazufloxacin. Additionally, a few 7-monoalkylaminomethyl derivatives exhibited good activities against both Gram-positive and Gram-negative organisms.     

Cytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in gram-positive bacteria

Type III secretion for injection of effector proteins into host cells has not been described for Gram-positive bacteria despite their importance in disease. Here, we describe an injection pathway for the Gram-positive pathogen Streptococcus pyogenes that utilizes streptolysin O (SLO), a cholesterol-dependent cytolysin. The data support a model in which an effector is translocated through the SLO pore by a polarized process. The effector, SPN (S. pyogenes NAD-glycohydrolase), is capable of producing the potent second messenger cyclic ADP-ribose, and SLO and SPN act synergistically to trigger cytotoxicity. These data provide a novel paradigm for the function of the cholesterol-dependent cytolysin family and its wide distribution suggests that cytolysin-mediated translocation (CMT) may be the equivalent of type III secretion for Gram-positive pathogens.     

A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA.

Choline binding proteins are virulence determinants present in several Gram-positive bacteria. Because anchorage of these proteins to the cell wall through their choline binding domain is essential for bacterial virulence, their release from the cell surface is considered a powerful target for a weapon against these pathogens. The first crystal structure of a choline binding domain, from the toxin-releasing enzyme pneumococcal major autolysin (LytA), reveals a novel solenoid fold consisting exclusively of beta-hairpins that stack to form a left-handed superhelix. This unique structure is maintained by choline molecules at the hydrophobic interface of consecutive hairpins and may be present in other choline binding proteins that share high homology to the repeated motif of the domain.     

Virtual screening of LPXTG competitive SrtA inhibitors targeting signal transduction mechanism in Bacillus anthracis: a combined experimental and theoretical study

Abstract

Members of the sortase enzyme super family decorate the surfaces of Bacillus anthracis cell wall with proteins that play key roles in microbial pathogenesis and its biofilm formation. Bacillus anthracis Sortase-A (Ba-SrtA) is a potential target for new therapeutics as it is required for B. anthracis survival and replication within macrophages. An understanding of the binding site pocket and substrate recognition mechanism by SrtA enzymes may serve to be beneficial in the rational development of sortase inhibitors. Here, the LPXTG signal peptide-based competitive inhibitors are screened against the Ba-SrtA and compounds with reasonable inhibition, specificity, and mechanisms of inactivation of SrtA have been covered. The screened compounds are experimentally validated against the phylogenetically similar Gram-positive pathogen B. cereus. In situ microscopic visualizations suggest that these screened compounds showed the microbial and biofilm inhibitory activity against B. cereus. It facilitates the further development of these molecules into useful anti-infective agents to treat infections caused by B. anthracis and other Gram-positive pathogens. These results provide insight into basic design principles for generating new clinically relevant lead molecules. It also provides an alternative strategy where a screened ligand molecule can be used in combination to battle increasingly against the Gram-positive pathogens.     

Pili with strong attachments: Gram-positive bacteria do it differently

Bacteria attach to their appropriate environmental niche by using adhesins. To maximize their contact with the environment, adhesins are often present on the ends of long hairlike structures called pili. Recently, attention has focused on pili of Gram-positive bacteria because they may be vaccine candidates in important human pathogens. These pili differ from the well-studied pili of Gram-negative bacteria because their subunits are covalently linked, they do not require specific chaperones for assembly, and the tip protein (likely to be the adhesin) is not required to initiate formation of the pilus structure. In Gram-positive bacteria, the genes for pili occur in clusters, which may constitute mobile genetic elements. These clusters include the transpeptidase(s) of the sortase family that is/are required for polymerization of the subunit proteins. However, efficient covalent attachment of the completed pilus structure to the cell wall is accomplished, in cases where this has been studied, by the 'housekeeping' sortase, which is responsible for attachment to the peptidoglycan of most surface proteins containing cell wall sorting signals. This enzyme is encoded elsewhere on the genome. Because pili of Gram-positive bacteria have not been extensively investigated yet, we hope that this MicroReview will help to pinpoint the areas most in need of further study.     

Bacillus anthracis secretes proteins that mediate heme acquisition from hemoglobin

Acquisition of iron is necessary for the replication of nearly all bacterial pathogens; however, iron of vertebrate hosts is mostly sequestered by heme and bound to hemoglobin within red blood cells. In Bacillus anthracis, the spore-forming agent of anthrax, the mechanisms of iron scavenging from hemoglobin are unknown. We report here that B. anthracis secretes IsdX1 and IsdX2, two NEAT domain proteins, to remove heme from hemoglobin, thereby retrieving iron for bacterial growth. Unlike other Gram-positive bacteria, which rely on cell wall anchored Isd proteins for heme scavenging, B. anthracis seems to have also evolved NEAT domain proteins in the extracellular milieu and in the bacterial envelope to provide for the passage of heme.     

Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA

Genetic evidence indicates that Drosophila defense against Gram-positive bacteria is mediated by two putative pattern recognition receptors acting upstream of Toll, namely Gram-negative binding protein 1 (GNBP1) and peptidoglycan recognition protein SA (PGRP-SA). Until now however, the molecular recognition proceedings for sensing of Gram-positive pathogens were not known. In the present, we report the physical interaction between GNBP1 and PGRP-SA using recombinant proteins. GNBP1 was able to hydrolyze Gram-positive peptidoglycan (PG), while PGRP-SA bound highly purified PG fragments (muropeptides). Interaction between these proteins was enhanced in the presence of PG or muropeptides. PGRP-SA binding depended on the polymerization status of the muropeptides, pointing to constraints in the number of PGRP-SA molecules bound for signaling initiation. We propose a model whereby GNBP1 presents a processed form of PG for sensing by PGRP-SA and that a tripartite interaction between these proteins and PG is essential for downstream signaling.     

The role of Streptococcus pneumoniae sortase A in colonisation and pathogenesis

Sortase enzymes are found throughout Gram-positive bacteria and are responsible for the covalent attachment of specific proteins to the cell wall. Through the anchoring of these cell wall proteins, sortase enzymes are important in the ability of several Gram-positive pathogens to cause disease. Previously, deletion of srtA from Streptococcus pneumoniae (the pneumococcus) was shown to disturb the localisation of surface proteins, and decrease bacterial adherence to human pharyngeal cells in vitro. Here we present data demonstrating, for the first time, a role for srtA as a pneumococcal fitness factor in experimental models of pneumonia and bacteraemia. In addition, srtA contributed to nasopharyngeal colonisation in vivo. Furthermore, we find that the contribution of srtA varied between two pneumococcal strains. We show that the known role of srtA in adherence in vitro is dependent on capsule expression, the role of SrtA in adherence to human cells only being apparent in the absence of the pneumococcal capsule. The srtA gene was detected by PCR in all 82 clinical isolates examined and sequencing of the gene from 20 strains showed srtA to be highly conserved. The ubiquitous distribution of srtA was in contrast to the other known pneumococcal sortase genes, srtB, C and D, which were found in only 14 of the 82 tested strains (17%).     

What are archaebacteria: life's third domain or monoderm prokaryotes related to Gram-positive bacteria? A new proposal for the classification of prokaryotic organisms

The evolutionary relationship within prokaryotes is examined based on signature sequences (defined as conserved inserts or deletions shared by specific taxa) and phylogenies derived from different proteins. Archaebacteria are indicated as being monophyletic by a number of proteins related to the information transfer processes. In contrast, for several other highly conserved proteins, common signature sequences are present in archaebacteria and Gram-positive bacteria, whereas Gram-negative bacteria are indicated as being distinct. For these proteins, archaebacteria do not form a phylogenetically distinct clade but show polyphyletic branching within Gram-positive bacteria. A closer relationship of archaebacteria to Gram-positive bacteria in comparison with Gram-negative bacteria is generally seen for the majority of the available gene/protein sequences. To account for these results and the fact that both archaebacteria and Gram-positive bacteria are prokaryotes surrounded by a single cell membrane, I propose that the primary division within prokaryotes is between monoderm prokaryotes (surrounded by a single membrane) and diderm prokaryotes (i.e. all true Gram-negative bacteria containing both an inner cytoplasmic membrane and an outer membrane). This proposal is consistent with both cell morphology and signature sequences in different proteins. The monophyletic nature of archaebacteria for some genes, and their polyphyletic branching within Gram-positive bacteria as suggested by others, is critically examined, and several explanations, including derivation of archaebacteria from Gram-positive bacteria in response to antibiotic selection pressure, are proposed. Signature sequences in proteins also indicate that the low-G + C Gram-positive bacteria are phylogenetically distinct from the high-G + C Gram-positive group and that the diderm prokaryotes (i.e. Gram-negative bacteria) appear to have evolved from the latter group. Protein phylogenies and signature sequences also show that all eukaryotic cells have received significant gene contributions from both an archaebacterium and a Gram-negative eubacterium. Thus, the hypothesis that archaebacteria and eukaryotes shared a common ancestor exclusive of eubacteria is not supported. These observations provide evidence for an alternate view of the evolutionary relationship among living organisms that is different from the currently popular three-domain proposal.     

The plant-derived chalcone Xanthoangelol targets the membrane of Gram-positive bacteria

Xanthoangelol is a geranylated chalcone isolated from fruits of Amorpha fructicosa that exhibits antibacterial effects at low micromolar concentration against Gram-positive bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium and Enterococcus faecalis. We demonstrate that Xanthoangelol treatment of Gram-positive bacteria affects bacterial membrane integrity and leads to a leakage of intracellular metabolites. This correlates with a rapid collapse of the membrane potential and results in a fast and strong bactericidal effect. Proteomic profiling of Xanthoangelol-treated cells revealed signatures of cell wall and/or membrane damage and oxidative stress. Xanthoangelol specifically disturbs the membrane of Gram-positive bacteria potentially by forming pores resulting in cell lysis. In contrast, Xanthoangelol treatment of human cells showed only mildly hemolytic and cytotoxic effects at higher concentrations. Therefore, geranylated chalcones such as Xanthoangelol are promising lead structures for new antimicrobials against drug-resistant gram-positive pathogens.