Exo‐ and surface proteomes of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM is a well‐known probiotic bacterium extensively studied for its beneficial health effects. Exoproteome (proteins exported into culture medium) and surface proteome (proteins attached to S‐layer) of this probiotic were identified by using 2DE followed by MALDI TOF MS to find proteins potentially involved in bacteria–host interactions. The exo‐ and surface proteomes included 43 and 39 different proteins from 72 and 49 successfully identified spots, respectively. Twenty‐two proteins were shared between the two proteomes; both contained the major surface layer protein that participates in host interaction as well as several well‐known and putative moonlighting proteins. The exoproteome contained nine classically‐secreted (containing a signal sequence) and ten nonclassically‐secreted proteins, while the surface proteome contained four classically‐secreted and eight nonclassically secreted proteins. Identification of exo‐ and surface proteomes contributes describing potential protein‐mediated probiotic–host interactions.     

An extracellular Leptospira interrogans leucine‐rich repeat protein binds human E‐ and VE‐cadherins

Pathogenic Leptospira bacteria are the causative agents of leptospirosis, a zoonotic disease affecting animals and humans worldwide. These pathogenic species have the ability to rapidly cross host tissue barriers by a yet unknown mechanism. A comparative analysis of pathogens and saprophytes revealed a higher abundance of genes encoding proteins with leucine‐rich repeat (LRR) domains in the genomes of pathogens. In other bacterial pathogens, proteins with LRR domains have been shown to be involved in mediating host cell attachment and invasion. One protein from the pathogenic species Leptospira interrogans, LIC10831, has been previously analysed via X‐ray crystallography, with findings suggesting it may be an important bacterial adhesin. Herein we show that LIC10831 elicits an antibody response in infected animals, is actively secreted by the bacterium, and binds human E‐ and VE‐cadherins. These results provide biochemical and cellular evidences of LRR protein‐mediated host–pathogen interactions and identify a new multireceptor binding protein from this infectious Leptospira species.     

Mammalian cell entry (Mce) protein of Leptospira interrogans binds extracellular matrix components,plasminogen and β2 integrin

A severe re‐emergingzoonosis, leptospirosis, is caused by pathogenic spirochetes of the genus Leptospira. Several studies have identified leptospiral surface proteins with the ability to bind ECM and plasma components, which could mediate adhesion and invasion through the hosts. It has been shown that Mce of pathogenic Leptospira spp. is an RGD (Arg‐Gly‐Asp)‐motif‐dependent virulence factor, responsible for infection of cells and animals. In the present article, we decided to further study the repertoire of the Mce activities in leptospiral biological properties. We report that the recombinant Mce is a broad‐spectrum ECM‐binding protein, capable of interacting with laminin, cellular and plasma fibronectin and collagen IV. Dose­–r­esponse interaction was observed for all the components, fulfilling ligand­–receptor requirements. Mce is a PLG binding protein capable to recruit this component from NHS, generating PLA in the presence of PLG activator. Binding of Mce was also observed with the leukocyte cell receptors αLβ2 [(CD11a/CD18)‐LFA‐1] and αMβ2 [(CD11b/CD18)‐Mac‐1], suggesting the involvement of this protein in the host immune response. Indeed, virulent Leptospira L1‐130 was capable of binding both integrins, whereas culture‐attenuated M‐20 strain only bind to αMβ2 [(CD11b/CD18)‐Mac‐1]. To the best of our knowledge, this is the first work to describe that Mce surface protein could mediate the attachment of Leptospira interrogans to human cell receptors αLβ2(CD11a/CD18) and αMβ2(CD11b/CD18).     

乙酰化修饰在嗜肺军团菌致病过程中的作用

乙酰化修饰是由乙酰基转移酶、去乙酰化酶介导的可逆的蛋白质翻译后修饰。其中,乙酰基转移酶将乙酰辅酶A的乙酰基团转移至底物蛋白的氨基酸残基,而乙酰基团的去除由去乙酰化酶完成。乙酰化修饰参与许多基本生物学过程的调节作用,越来越多的研究表明,蛋白质乙酰化修饰在病原菌的致病过程中具有重要作用。病原菌,如引起非典型性肺炎的嗜肺军团菌,可以通过分泌具有乙酰基转移酶活性的效应蛋白靶向宿主细胞信号通路的关键蛋白质因子,干扰宿主细胞信号通路及免疫反应。本文主要从嗜肺军团菌的致病机制、乙酰化修饰及乙酰化修饰在病原体致病过程中的调控作用进行综述,突出已知的乙酰化毒力蛋白的例子,并讨论它们如何影响与宿主的相互作用,为理解乙酰化修饰在嗜肺军团菌致病过程中的作用机制提供参考。     

Role of distinct type‐IV‐secretion systems and secreted effector sets in host adaptation by pathogenic Bartonella species

The α‐proteobacterial genus Bartonella comprises a large number of facultative intracellular pathogens that share a common lifestyle hallmarked by hemotrophic infection and arthropod transmission. Speciation in the four deep‐branching lineages (L1–L4) occurred by host adaptation facilitating the establishment of long lasting bacteraemia in specific mammalian reservoir host(s). Two distinct type‐IV‐secretion systems (T4SSs) acquired horizontally by different Bartonella lineages mediate essential host interactions during infection and represent key innovations for host adaptation. The Trw‐T4SS confined to the species‐rich L4 mediates host‐specific erythrocyte infection and likely has functionally replaced flagella as ancestral virulence factors implicated in erythrocyte colonisation by bartonellae of the other lineages. The VirB/VirD4‐T4SS translocates Bartonella effector proteins (Bep) into various host cell types to modulate diverse cellular and innate immune functions involved in systemic spreading of bacteria following intradermal inoculation. Independent acquisition of the virB/virD4/bep locus by L1, L3, and L4 was likely driven by arthropod vectors associated with intradermal inoculation of bacteria rather than facilitating direct access to blood. Subsequently, adaptation to colonise specific niches in the new host has shaped the evolution of complex species‐specific Bep repertoires. This diversification of the virulence factor repertoire of Bartonella spp. represents a remarkable example for parallel evolution of host adaptation.     

Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector,phyllogen, induces phyllody

Plant pathogens alter the course of plant developmental processes, resulting in abnormal morphology in infected host plants. Phytoplasmas are unique plant‐pathogenic bacteria that transform plant floral organs into leaf‐like structures and cause the emergence of secondary flowers. These distinctive symptoms have attracted considerable interest for many years. Here, we revealed the molecular mechanisms of the floral symptoms by focusing on a phytoplasma‐secreted protein, PHYL1, which induces morphological changes in flowers that are similar to those seen in phytoplasma‐infected plants. PHYL1 is a homolog of the phytoplasmal effector SAP54 that also alters floral development. Using yeast two‐hybrid and in planta transient co‐expression assays, we found that PHYL1 interacts with and degrades the floral homeotic MADS domain proteins SEPALLATA3 (SEP3), APETALA1 (AP1) and CAULIFLOWER (CAL). This degradation of MADS domain proteins was dependent on the ubiquitin–proteasome pathway. The expression of floral development genes downstream of SEP3 and AP1 was disrupted in 35S::PHYL1 transgenic plants. PHYL1 was genetically and functionally conserved among other phytoplasma strains and species. We designate PHYL1, SAP54 and their homologs as members of the phyllody‐inducing gene family of ‘phyllogens’.     

Borrelia burgdorferi locus BB0795 encodes a BamA orthologue required for growth and efficient localization of outer membrane proteins

The outer membrane (OM) of the pathogenic diderm spirochete, Borrelia burgdorferi, contains integral β‐barrel outer membrane proteins (OMPs) in addition to its numerous outer surface lipoproteins. Very few OMPs have been identified in B. burgdorferi, and the protein machinery required for OMP assembly and OM localization is currently unknown. Essential OM BamA proteins have recently been characterized in Gram‐negative bacteria that are central components of an OM β‐barrel assembly machine and are required for proper localization and insertion of bacterial OMPs. In the present study, we characterized a putative B. burgdorferi BamA orthologue encoded by open reading frame bb0795. Structural model predictions and cellular localization data indicate that the B. burgdorferi BB0795 protein contains an N‐terminal periplasmic domain and a C‐terminal, surface‐exposed β‐barrel domain. Additionally, assays with an IPTG‐regulatable bb0795 mutant revealed that BB0795 is required for B. burgdorferi growth. Furthermore, depletion of BB0795 results in decreased amounts of detectable OMPs in the B. burgdorferi OM. Interestingly, a decrease in the levels of surface‐exposed lipoproteins was also observed in the mutant OMs. Collectively, our structural, cellular localization and functional data are consistent with the characteristics of other BamA proteins, indicating that BB0795 is a B. burgdorferi BamA orthologue.     

Global proteomic pattern of Tropheryma whipplei: A Whipple's disease bacterium

The proteome of Tropheryma whipplei, the intracellular bacterium responsible for Whipple's disease (WD), was analyzed using two complementary approaches: 2‐DE coupled with MALDI‐TOF and SDS‐PAGE with nanoLC‐MS/MS. This strategy led to the identification of 206 proteins of 808 predicted ORFs, resolving some questions raised by the genomic sequence of this bacterium. We successfully identified antibiotic targets and proteins with predicted N‐terminal signal sequences. Additionally, we identified a family of surface proteins (known as T. whipplei surface proteins (WiSPs)), which are encoded by a unique group of species‐specific genes and serve as both coding regions and DNA repeats that promote genomic recombination. Comparison of the protein expression profiles of the intracellular facultative host‐associated WD bacterium with other host‐associated, intracellular obligate, and environmental bacteria revealed that T. whipplei shares a proteomic expression profile with other host‐associated facultative intracellular bacteria. In summary, this study describes the global protein expression pattern of T. whipplei and reveals some specific features of the T. whipplei proteome.     

Flagellin phase‐dependent swimming on epithelial cell surfaces contributes to productive Salmonella gut colonisation

The flagellum is a sophisticated nanomachine and an important virulence factor of many pathogenic bacteria. Flagellar motility enables directed movements towards host cells in a chemotactic process, and near‐surface swimming on cell surfaces is crucial for selection of permissive entry sites. The long external flagellar filament is made of tens of thousands subunits of a single protein, flagellin, and many Salmonella serovars alternate expression of antigenically distinct flagellin proteins, FliC and FljB. However, the role of the different flagellin variants during gut colonisation and host cell invasion remains elusive. Here, we demonstrate that flagella made of different flagellin variants display structural differences and affect Salmonella's swimming behaviour on host cell surfaces. We observed a distinct advantage of bacteria expressing FliC‐flagella to identify target sites on host cell surfaces and to invade epithelial cells. FliC‐expressing bacteria outcompeted FljB‐expressing bacteria for intestinal tissue colonisation in the gastroenteritis and typhoid murine infection models. Intracellular survival and responses of the host immune system were not altered. We conclude that structural properties of flagella modulate the swimming behaviour on host cell surfaces, which facilitates the search for invasion sites and might constitute a general mechanism for productive host cell invasion of flagellated bacteria.     

Solution structure of a bacterial immunoglobulin‐like domain of the outer membrane protein (LigB) from Leptospira

Leptospiral immunoglobulin‐like (Lig) proteins are surface proteins expressed in pathogenic strains of Leptospira. LigB, an outer membrane protein containing tandem repeats of bacterial Ig‐like (Big) domains and a no‐repeat tail, has been identified as a virulence factor involved in adhesion of pathogenic Leptospira interrogans to host cells. A Big domain of LigB, LigBCen2R, was reported previously to bind the GBD domain of fibronectin, suggesting its important role in leptospiral infections. In this study, we determined the solution structure of LigBCen2R by nuclear magnetic resonance (NMR) spectroscopy. LigBCen2R adopts a canonical immunoglobulin‐like fold which is comprised of a beta‐sandwich of ten strands in three sheets. We indicated that LigBCen2R is able to bind to Ca²⁺ with a high affinity by isothermal titration calorimetry assay. NMR perturbation experiment identified a number of residues responsible for Ca²⁺ binding. Structural comparison of it with other Big domains demonstrates that they share a similar fold pattern, but vary in some structural characters. Since Lig proteins play a vital role in the infection to host cells, our study will contribute a structural basis to understand the interactions between Leptospira and host cells. Proteins 2015; 83:195–200. © 2014 Wiley Periodicals, Inc.     

Staphylococcus aureus proteins SSL6 and SElX interact with neutrophil receptors as identified using secretome phage display

In order to cause colonization and invasive disease, pathogenic bacteria secrete proteins that modulate host immune defences. Identification and characterization of these proteins leads to a better understanding of the pathological processes underlying infectious and inflammatory diseases and is essential in the development of new strategies for their prevention and treatment. Current techniques to functionally characterize these proteins are laborious and inefficient. Here we describe a high‐throughput functional selection strategy using phage display in order to identify immune evasion proteins. Using this technique we identified two previously uncharacterized proteins secreted by Staphylococcus aureus, SElX and SSL6 that bind to neutrophil surface receptors. SElX binds PSGL‐1 on neutrophils and thereby inhibits the interaction between PSGL‐1 and P‐selectin, a crucial step in the recruitment of neutrophils to the site of infection. SSL6 is the first bacterial protein identified that binds CD47, a widely expressed cell surface protein recently described as an interesting target in anti‐cancer therapy. Our findings provide new insights into the pathogenesis of S. aureus infections and support phage display as an efficient method to identify bacterial secretome proteins interacting with humoral or cellular immune components.     

Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages

Sphingomyelinases secreted by pathogenic bacteria play important roles in host–pathogen interactions ranging from interfering with phagocytosis and oxidative burst to iron acquisition. This study shows that the Mtb protein Rv0888 possesses potent sphingomyelinase activity cleaving sphingomyelin, a major lipid in eukaryotic cells, into ceramide and phosphocholine, which are then utilized by Mtb as carbon, nitrogen and phosphorus sources, respectively. An Mtb rv0888 deletion mutant did not grow on sphingomyelin as a sole carbon source anymore and replicated poorly in macrophages indicating that Mtb utilizes sphingomyelin during infection. Rv0888 is an unusual membrane protein with a surface‐exposed C‐terminal sphingomyelinase domain and a putative N‐terminal channel domain that mediated glucose and phosphocholine uptake across the outer membrane in an M. smegmatis porin mutant. Hence, we propose to name Rv0888 as SpmT (sp hingomyelinase of M ycobacterium t uberculosis). Erythrocyte membranes contain up to 27% sphingomyelin. The finding that Rv0888 accounts for half of Mtb's hemolytic activity is consistent with its sphingomyelinase activity and the observation that Rv0888 levels are increased in the presence of erythrocytes and sphingomyelin by 5‐ and 100‐fold, respectively. Thus, Rv0888 is a novel outer membrane protein that enables Mtb to utilize sphingomyelin as a source of several essential nutrients during intracellular growth.     

Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false‐positive control

Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface‐exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital‐acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface “shaving” technique with either trypsin or proteinase‐K combined with LC‐MS/MS. Trypsin‐derived data were controlled using a “false‐positive” strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell lysis and were removed from the trypsin‐shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface‐exposed peptides. Trypsin and proteinase‐K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase‐K treatment, 13 specific to trypsin and three identified in the control. The use of a subtracted false‐positive strategy improved enrichment of surface‐exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance–surface protein SACOL0050 (pls) and penicillin‐binding protein 2′ (mecA), as well as bifunctional autolysin and the extracellular matrix‐binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface‐exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus.     

A dual role for proline iminopeptidase in the regulation of bacterial motility and host immunity

During plant–pathogen interactions, pathogenic bacteria have evolved multiple strategies to cope with the sophisticated defence systems of host plants. Proline iminopeptidase (PIP) is essential to Xanthomonas campestris pv. campestris (Xcc) virulence, and is conserved in many plant‐associated bacteria, but its pathogenic mechanism remains unclear. In this study, we found that disruption of pip in Xcc enhanced its flagella‐mediated bacterial motility by decreasing intracellular bis‐(3′,5′)‐cyclic dimeric guanosine monophosphate (c‐di‐GMP) levels, whereas overexpression of pip in Xcc restricted its bacterial motility by elevating c‐di‐GMP levels. We also found that PIP is a type III secretion system‐dependent effector capable of eliciting a hypersensitive response in non‐host, but not host plants. When we transformed pip into the host plant Arabidopsis, higher bacterial titres were observed in pip‐overexpressing plants relative to wild‐type plants after Xcc inoculation. The repressive function of PIP on plant immunity was dependent on PIP's enzymatic activity and acted through interference with the salicylic acid (SA) biosynthetic and regulatory genes. Thus, PIP simultaneously regulates two distinct regulatory networks during plant–microbe interactions, i.e. it affects intracellular c‐di‐GMP levels to coordinate bacterial behaviour, such as motility, and functions as a type III effector translocated into plant cells to suppress plant immunity. Both processes provide bacteria with the regulatory potential to rapidly adapt to complex environments, to utilize limited resources for growth and survival in a cost‐efficient manner and to improve the chances of bacterial survival by helping pathogens to inhabit the internal tissues of host plants.     

Burkholderia BcpA mediates biofilm formation independently of interbacterial contact‐dependent growth inhibition

Contact‐dependent growth inhibition (CDI) is a phenomenon in which Gram‐negative bacteria use the toxic C‐terminus of a large surface‐exposed exoprotein to inhibit the growth of susceptible bacteria upon cell–cell contact. Little is known about when and where bacteria express the genes encoding CDI system proteins and how these systems contribute to the survival of bacteria in their natural niche. Here we establish that, in addition to mediating interbacterial competition, the Burkholderia thailandensis CDI system exoprotein BcpA is required for biofilm development. We also provide evidence that the catalytic activity of BcpA and extracellular DNA are required for the characteristic biofilm pillars to form. We show using a bcpA–gfp fusion that within the biofilm, expression of the CDI system‐encoding genes is below the limit of detection for the majority of bacteria and only a subset of cells express the genes strongly at any given time. Analysis of a strain constitutively expressing the genes indicates that native expression is critical for biofilm architecture. Although CDI systems have so far only been demonstrated to be involved in interbacterial competition, constitutive production of the system's immunity protein in the entire bacterial population did not alter biofilm formation, indicating a CDI‐independent role for BcpA in this process. We propose, therefore, that bacteria may use CDI proteins in cooperative behaviours, like building biofilm communities, and in competitive behaviours that prevent non‐self bacteria from entering the community.     

BigA is a novel adhesin of Brucella that mediates adhesion to epithelial cells

Adhesion to cells is the initial step in the infectious cycle of basically all pathogenic bacteria, and to do so, microorganisms have evolved surface molecules that target different cellular receptors. Brucella is an intracellular pathogen that infects a wide range of mammals whose virulence is completely dependent on the capacity to replicate in phagocytes. Although much has been done to elucidate how Brucella multiplies in macrophages, we still do not understand how bacteria invade epithelial cells to perform a replicative cycle or what adhesion molecules are involved in the process. We report the identification in Brucella abortus of a novel adhesin that harbours a bacterial immunoglobulin‐like domain and demonstrate that this protein is involved in the adhesion to polarized epithelial cells such as the Caco‐2 and Madin–Darby canine kidney models targeting the bacteria to the cell–cell interaction membrane. While deletion of the gene significantly reduced adhesion, over‐expression dramatically increased it. Addition of the recombinant protein to cells induced cytoskeleton rearrangements and showed that this adhesin targets proteins of the cell–cell interaction membrane in confluent cultures.     

Leptospira: a spirochaete with a hybrid outer membrane

Leptospira is a genus of spirochaetes that includes organisms with a variety of lifestyles ranging from aquatic saprophytes to invasive pathogens. Adaptation to a wide variety of environmental conditions has required leptospires to acquire a large genome and a complex outer membrane with features that are unique among bacteria. The most abundant surface‐exposed outer membrane proteins are lipoproteins that are integrated into the lipid bilayer by amino‐terminal fatty acids. In contrast to many spirochaetes, the leptospiral outer membrane also includes lipopolysaccharide and many homologues of well‐known beta‐barrel transmembrane outer membrane proteins. Research on leptospiral transmembrane outer membrane proteins has lagged behind studies of lipoproteins because of their aberrant behaviour by Triton X‐114 detergent fractionation. For this reason, transmembrane outer membrane proteins are best characterized by assessing membrane integration and surface exposure. Not surprisingly, some outer membrane proteins that mediate host–pathogen interactions are strongly regulated by conditions found in mammalian host tissues. For example, the leptospiral immunoglobulin‐like (Lig) repeat proteins are dramatically induced by osmolarity and mediate interactions with host extracellular matrix proteins. Development of molecular genetic tools are making it possible to finally understand the roles of these and other outer membrane proteins in mechanisms of leptospiral pathogenesis.