Screening a library of temperature-sensitive mutants to identify secretion factors in .

Protein secretion is an essential cell process in bacteria, required for cell envelope biogenesis, export of virulence factors, and acquisition of nutrients, among other important functions. In the Sec secretion pathway, signal peptide-bearing precursors are recognized by the SecA ATPase and pushed across the membrane through a translocon channel made of the proteins SecY, SecE, and SecG. The Sec pathway has been extensively studied in the model organism , but the Sec pathways of other bacteria such as the human pathogen differ in important ways from this model.

Deficiency in non-classical major histocompatibility class II-like molecule, H2-O confers protection against Staphylococcus aureus in mice.

Staphylococcus aureus is a human-adapted pathogen that replicates by asymptomatically colonizing its host. S. aureus is also the causative agent of purulent skin and soft tissue infections as well as bloodstream infections that result in the metastatic seeding of abscess lesions in all organ tissues.

Processing of LtaS restricts LTA assembly and YSIRK preprotein trafficking into cross-walls.

Septal membranes of serve as the site of secretion for precursors endowed with the YSIRK motif. Depletion of , a gene required for lipoteichoic acid (LTA) synthesis, results in the loss of restricted trafficking of YSIRK precursors to septal membranes. Here, we seek to understand the mechanism that ties LTA assembly and trafficking of YSIRK precursors.

Immunoglobulin G subclasses confer protection against bloodstream dissemination through distinct mechanisms in mouse models.

Antibodies bind target molecules with exquisite specificity. The removal of these targets is mediated by the effector functions of antibodies. We reported earlier that the monoclonal antibody (mAb) 3F6 promotes opsonophagocytic killing of in blood and reduces bacterial replication in animals.

Modeling gastrointestinal anthrax disease.

Bacillus anthracis is a spore-forming microbe that persists in soil and causes anthrax disease. The most natural route of infection is ingestion by grazing animals. Gastrointestinal (GI) anthrax also occurs in their monogastric predators, including humans.

Toward Optimization of a Rabbit Model of Staphylococcus aureus (USA300) Skin and Soft Tissue Infection.

Staphylococcus aureus remains a leading cause of skin and soft tissue infections (SSTIs) globally. In the United States, many of these infections are caused by isolates classified as USA300. Our understanding of the success of USA300 as a human pathogen is due in part to data obtained from animal infection models, including rabbit SSTI models.

Engineered human antibodies for the opsonization and killing of .

Gram-positive organisms with their thick envelope cannot be lysed by complement alone. Nonetheless, antibody-binding on the surface can recruit complement and mark these invaders for uptake and killing by phagocytes, a process known as opsonophagocytosis. The crystallizable fragment of immunoglobulins (Fcγ) is key for complement recruitment.

A protein A based Staphylococcus aureus vaccine with improved safety.

Exposure to Staphylococcus aureus does not lead to immunity as evidenced by the persistent colonization of one third of the human population. S. aureus immune escape is mediated by factors that preempt complement activation, destroy phagocytes, and modify B and T cell responses.

Peptidoglycan Contribution to the B Cell Superantigen Activity of Staphylococcal Protein A.

causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of display the abundant staphylococcal protein A (SpA).

Regulated cleavage of glycan strands by the murein hydrolase SagB in involves a direct interaction with LyrA (SpdC).

LyrA (SpdC), a homologue of eukaryotic CAAX proteases that act on prenylated substrates, has been implicated in the assembly of several pathways of the envelope of We described earlier the sostaphin esistance (Lyr) and taphylococcal rotein A isplay (Spd) phenotypes associated with loss of the () gene. However, a direct contribution to the assembly of pentaglycine crossbridges, the target of lysostaphin cleavage in peptidoglycan, or of Staphylococcal protein A attachment to peptidoglycan could not be attributed directly to LyrA (SpdC). These two processes are catalyzed by the Fem factors and Sortase A, respectively.

The Expression of von Willebrand Factor-Binding Protein Determines Joint-Invading Capacity of Staphylococcus aureus, a Core Mechanism of Septic Arthritis.

Septic arthritis, one of the most dangerous joint diseases, is predominantly caused by In contrast, coagulase-negative staphylococci are rarely found in septic arthritis. We hypothesize that coagulases released by , including coagulase (Coa) and von Willebrand factor-binding protein (vWbp), play potent roles in the induction of septic arthritis. Four isogenic strains differing in expression of coagulases (wild-type [WT] Newman, Δ, Δ, and Δ Δ) were used to induce septic arthritis in both wild-type and von Willebrand factor (vWF)-deficient mice.

Glycosylation-dependent opsonophagocytic activity of staphylococcal protein A antibodies.

Antibodies may bind to bacterial pathogens or their toxins to control infections, and their effector activity is mediated through the recruitment of complement component C1q or the engagement with Fcγ receptors (FcγRs). For bacterial pathogens that rely on a single toxin to cause disease, immunity correlates with toxin neutralization. Most other bacterial pathogens, including , secrete numerous toxins and evolved multiple mechanisms to escape opsonization and complement killing.

FmhA and FmhC of incorporate serine residues into peptidoglycan cross-bridges.

Staphylococcal peptidoglycan is characterized by pentaglycine cross-bridges that are cross-linked between adjacent wall peptides by penicillin-binding proteins to confer robustness and flexibility. In , pentaglycine cross-bridges are synthesized by three proteins: FemX adds the first glycine, and the homodimers FemA and FemB sequentially add two Gly-Gly dipeptides. Occasionally, serine residues are also incorporated into the cross-bridges by enzymes that have heretofore not been identified.

Distinct Pathways Carry Out α and β Galactosylation of Secondary Cell Wall Polysaccharide in Bacillus anthracis.

, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is required for the retention of surface layer (S-layer) and S-layer homology (SLH) domain proteins. Genetic disruption of the SCWP biosynthetic pathway impairs growth and cell division. SCWP is comprised of trisaccharide repeats composed of one ManNAc and two GlcNAc residues with O-3-α-Gal and O-4-β-Gal substitutions.

Rapid pathogen identification and antimicrobial susceptibility testing in in vitro endophthalmitis with matrix assisted laser desorption-ionization Time-of-Flight Mass Spectrometry and VITEK 2 without prior culture.

Purpose: Prompt clinical diagnosis and initiation of treatment are critical in the management of infectious endophthalmitis. Current methods used to identify causative agents of infectious endophthalmitis are mostly inefficient, owing to suboptimal sensitivity, length, and cost. Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) can be used to rapidly identity pathogens without a need for culture.

Staphylococcus aureus Exploits the Host Apoptotic Pathway To Persist during Infection.

is a deadly pathogen that causes fatal diseases in humans. During infection, secretes nuclease (Nuc) and adenosine synthase A (AdsA) to generate cytotoxic deoxyadenosine (dAdo) from neutrophil extracellular traps which triggers noninflammatory apoptosis in macrophages. In this manner, replicating staphylococci escape phagocytic killing without alerting the immune system.

FPR1 is the plague receptor on host immune cells.

The causative agent of plague, Yersinia pestis, uses a type III secretion system to selectively destroy immune cells in humans, thus enabling Y. pestis to reproduce in the bloodstream and be transmitted to new hosts through fleabites. The host factors that are responsible for the selective destruction of immune cells by plague bacteria are unknown.

B cell superantigens in the human intestinal microbiota.

IgA is prominently secreted at mucosal surfaces and coats a fraction of the commensal microbiota, a process that is critical for intestinal homeostasis. However, the mechanisms of IgA induction and the molecular targets of these antibodies remain poorly understood, particularly in humans. Here, we demonstrate that microbiota from a subset of human individuals encode two protein "superantigens" expressed on the surface of commensal bacteria of the family Lachnospiraceae such as that bind IgA variable regions and stimulate potent IgA responses in mice.

O antigen is the target of bactericidal Weil-Felix antibodies.

Rickettsial diseases have long been diagnosed with serum antibodies cross-reactive against (Weil-Felix reaction). Although Weil-Felix antibodies are associated with the development of immunity, their rickettsial target and contribution to disease pathogenesis are not established. Here, we developed a transposon for insertional mutagenesis of , isolating variants defective for replication in cultured cells and in spotted fever pathogenesis.

Staphylococcal Protein Secretion and Envelope Assembly.

The highly cross-linked peptidoglycan represents the rigid layer of the bacterial envelope and protects bacteria from osmotic lysis. In Gram-positive bacteria, peptidoglycan also functions as a scaffold for the immobilization of capsular polysaccharide, wall teichoic acid (WTA), and surface proteins. This chapter captures recent development on the assembly of the envelope of including mechanisms accounting for immobilization of molecules to peptidoglycan as well as hydrolysis of peptidoglycan for the specific release of bound molecules, facilitation of protein secretion across the envelope and cell division.

Staphylococcus aureus endocarditis: distinct mechanisms of bacterial adhesion to damaged and inflamed heart valves.

Aims: The pathogenesis of endocarditis is not well understood resulting in unsuccessful attempts at prevention. Clinical observations suggest that Staphylococcus aureus infects either damaged or inflamed heart valves. Using a newly developed endocarditis mouse model, we therefore studied the initial adhesion of S.

Extraction and Purification of Wall-Bound Polymers of Gram-Positive Bacteria.

The envelope of gram-positive bacteria encompasses the cell wall, a rigid exoskeleton comprised of peptidoglycan that provides protection against lysis and governs bacterial cell shapes. Peptidoglycan also serves as the site of attachment for proteins and nonproteinaceous polymers that interact with the bacterial environment. Nonproteinaceous molecules include teichoic acids, capsular polysaccharides, and secondary cell wall polysaccharides (SCWP).

Sortases, Surface Proteins, and Their Roles in Disease and Vaccine Development.

Sortases cleave short peptide motif sequences at the C-terminal end of secreted surface protein precursors and either attach these polypeptides to the peptidoglycan of Gram-positive bacteria or promote their assembly into pilus structures that are also attached to peptidoglycan. Sortase A, the enzyme first identified in the human pathogen , binds LPXTG motif sorting signals, cleaves between threonine (T) and glycine (G) residues, and forms an acyl enzyme between its active-site cysteine thiol and the carboxyl group of threonine (T). Sortase A acyl enzyme is relieved by the nucleophilic attack of the cross bridge amino group within lipid II, thereby generating surface protein linked to peptidoglycan precursor.

Staphylococcus aureus Decolonization of Mice With Monoclonal Antibody Neutralizing Protein A.

Staphylococcus aureus persistently colonizes the nasopharynx of about one-third of the human population, a key risk factor for community- and hospital-acquired invasive infections. Current strategies for S. aureus decolonization include topical and systemic administration of antibiotics, which is associated with selection for antibiotic resistance and posttreatment recolonization.