Penicillin-Binding Protein 2 Is Essential for the Integrity of Growing Cells of Escherichia coli ponB Strains

Analysis of Escherichia coli pbpA(Ts) or rodA(Ts) strains defective for penicillin-binding protein (PBP) 1A or PBP 1B indicated that the activity of PBP 2 is essential to prevent cell lysis in PBP 1B(-) strains and suggested that PBP 2 is active or activatable in rodA(Ts) mutants under restrictive conditions.     

Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg²⁺ or Ca²⁺ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg²⁺ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg²⁺ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg²⁺ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.     

Trypanosome Letm1 Protein Is Essential for Mitochondrial Potassium Homeostasis

Letm1 is a conserved protein in eukaryotes bearing energized mitochondria. Hemizygous deletion of its gene has been implicated in symptoms of the human disease Wolf-Hirschhorn syndrome. Studies almost exclusively performed in opisthokonts have attributed several roles to Letm1, including maintaining mitochondrial morphology, mediating either calcium or potassium/proton antiport, and facilitating mitochondrial translation. We address the ancestral function of Letm1 in the highly diverged protist and significant pathogen, Trypanosoma brucei. We demonstrate that Letm1 is involved in maintaining mitochondrial volume via potassium/proton exchange across the inner membrane. This role is essential in the vector-dwelling procyclic and mammal-infecting bloodstream stages as well as in Trypanosoma brucei evansi, a form of the latter stage lacking an organellar genome. In the pathogenic bloodstream stage, the mitochondrion consumes ATP to maintain an energized state, whereas that of T. brucei evansi also lacks a conventional proton-driven membrane potential. Thus, Letm1 performs its function in different physiological states, suggesting that ion homeostasis is among the few characterized essential pathways of the mitochondrion at this T. brucei life stage. Interestingly, Letm1 depletion in the procyclic stage can be complemented by exogenous expression of its human counterpart, highlighting the conservation of protein function between highly divergent species. Furthermore, although mitochondrial translation is affected upon Letm1 ablation, it is an indirect consequence of K⁺ accumulation in the matrix.     

Direct Quantitation of the Numbers of Individual Penicillin-Binding Proteins per Cell in Staphylococcus aureus

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in bacterial peptidoglycan assembly. The absolute numbers of each PBP were determined by direct measurement and have been reported for two Staphylococcus aureus strains, RN4220 (methicillin-sensitive S. aureus) and RN450M (methicillin-resistant S. aureus). From the specific activity of the labeled penicillin and the absolute number of disintegrations per minute, and from the number of CFU per milliliter calculated from proteins and optical density, a determination of the number of PBPs per cell was made. These numbers ranged from approximately 150 to 825 PBPs/cell and represent the first direct determination of absolute numbers of PBPs in S. aureus.     

Las1 Is an Essential Nuclear Protein Involved in Cell Morphogenesis and Cell Surface Growth

Saccharomyces cerevisiae mutations that cause a requirement for SSD1-v for viability were isolated, yielding one new gene, LAS1, and three previously identified genes, SIT4, BCK1/SLK1, and SMP3. Three of these genes, LAS1, SIT4, and BCK1/SLK1, encode proteins that have roles in bud formation or morphogenesis. LAS1 is essential and loss of LAS1 function causes the cells to arrest as 80% unbudded cells and 20% large budded cells that accumulate many vesicles at the mother-daughter neck. Overexpression of LAS1 results in extra cell surface projections in the mother cell, alterations in actin and SPA2 localization, and the accumulation of electron-dense structures along the periphery of both the mother cell and the bud. The nuclear localization of LAS1 suggests a role of LAS1 for regulating bud formation and morphogenesis via the expression of components that function directly in these processes.     

Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) with Antibodies against Synthetic Peptides Derived from Penicillin-Binding Protein 2′

Ten kinds of peptides (21 to 32 amino acids in length) were synthesized based on the reported amino acid sequences of the penicillin-binding protein 2′ (PBP2′) of methicillin-resistant Staphylococcus aureus (MRSA). Antibodies against these synthetic peptides (SPs) were generated by immunizing rabbits. The antibodies raised against all the peptides except for one reacted to PBP2′ of MRSA and to SPs used for immunization but not to any other protein of MRSA or methicillin-susceptible S. aureus (MSSA) tested by ELISA and Western blotting. A sandwich immunoradiometric assay (IRMA) for the detection of PBP2′ was developed using these antibodies. The method could detect PBP2′ extracted from as few as 3 × 10⁴ cells of a clinical MRSA isolate, and a good correlation between cell number and signal radio-count was observed. IRMA was positive for all 51 methicillin-resistant staphylococci isolated from patients, and was negative for all the 28 methicillin-susceptible ones and 19 strains of other bacterial species. IRMA could be a simple and reliable method for MRSA detection in the clinical bacterial laboratory.     

Protein A Mutants of Staphylococcus aureus

Staphylococcus aureus Cowan I was exposed to nitrosoguanidine or ethyl-methanesulfonate, and survivors were screened on nutrient agar plates containing rabbit anti-protein A serum for loss of protein A production. More than half of all protein A-deficient mutants also lacked nuclease, coagulase, alpha hemolysin, fibrinolysin, mannitol utilization, and the phage-type pattern. Mutants with a spectrum of these properties were also isolated. Induced or spontaneous reversions of the mutants were observed. The properties of the protein A-deficient mutants suggest that synthesis or release (or both) of a number of extracellular products of S. aureus is controlled by a common regulatory mechanism.     

Staphylococcus aureus Manganese Transport Protein C (MntC) Is an Extracellular Matrix- and Plasminogen-Binding Protein

Infections caused by Staphylococcus aureus – particularly nosocomial infections - represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM). Manganese transport protein C (MntC), a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA). The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation.     

The Giant Protein Ebh Is a Determinant of Staphylococcus aureus Cell Size and Complement Resistance

Staphylococcus aureus USA300, the clonal type associated with epidemic community-acquired methicillin-resistant S. aureus (MRSA) infections, displays the giant protein Ebh on its surface. Mutations that disrupt the ebh reading frame increase the volume of staphylococcal cells and alter the cross wall, a membrane-enclosed peptidoglycan synthesis and assembly compartment. S. aureusebh variants display increased sensitivity to oxacillin (methicillin) as well as susceptibility to complement-mediated killing. Mutations in ebh are associated with reduced survival of mutant staphylococci in blood and diminished virulence in mice. We propose that Ebh, following its secretion into the cross wall, contributes to the characteristic cell growth and envelope assembly pathways of S. aureus, thereby enabling complement resistance and the pathogenesis of staphylococcal infections.     

The Microtubule-Binding Protein Ensconsin Is an Essential Cofactor of Kinesin-1

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Highlights? Ensconsin is required for cargo transport driven by kinesin-1 ? The C terminus of ensconsin activates kinesin-1; its N terminus binds microtubules ? Kinesin-1 mutants without autoinhibitory activity do not require ensconsin     

Protein A-Mediated Multicellular Behavior in Staphylococcus aureus

The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.Staphylococcus aureus is a gram-positive bacterium that lives as part of the normal microflora on the skin and mucous membranes of humans and animals. If S. aureus passes through the epithelial barrier and reaches internal organs, it can cause a variety of diseases, ranging from minor skin infections, such as furuncles or boils, to severe infections, such as bacteremia, pneumonia, osteomyelitis, or endocarditis. Despite the progress with antibiotics in the treatment of bacterial infections over the last 2 decades, the number of infections due to S. aureus has increased (11, 30). The infection rate has been correlated with an increase in the use of prosthetic and indwelling devices in modern medical practices (24, 26). S. aureus, as well as other coagulase-negative staphylococci, displays a strong capacity to irreversibly attach to the surface of implanted medical devices and forms multilayered communities of bacteria, known as biofilms, that grow embedded in a self-produced extracellular matrix (23). The biofilm formation process occurs in two steps: first, bacterial cells irreversibly attach to a surface, and second, they interact with each other and accumulate in multilayered cell clusters embedded in a self-produced extracellular matrix. Primary attachment is mediated by physico-chemical cell surface properties as well as specific factors that mediate the attachment to the host-derived extracellular matrix components that rapidly coat the biomaterial following insertion into the patient. Numerous proteins from the MSCRAMMs family (microbial surface components recognizing adhesive matrix molecules) are involved in the first step of S. aureus biofilm formation, such as clumping factors ClfA (37) and ClfB (41) and fibrinogen and fibronectin binding proteins (FnBPA and FnBPB) (25, 31). Once bacteria accumulate in multilayered cell clusters, most have no direct contact with the surface, and thus cell-to-cell interactions become essential for biofilm development and maintenance. An extracellular polysaccharide intercellular adhesin (PIA, or PNAG), produced by icaADBC operon-encoded enzymes, is currently the best-characterized element mediating intercellular interactions in vitro (8, 23, 34, 35, 38). Alternatively, a number of surface proteins can replace PIA/PNAG exopolysaccharide in promoting intercellular adhesion and biofilm development, including the surface protein Bap (9). All the tested staphylococcal isolates harboring the bap gene were shown to be strong biofilm producers, and inactivation of the icaADBC operon in bap-positive strains had no effect on in vitro biofilm formation (57). Remarkably, proteins homologous to Bap are involved in the biofilm formation process in diverse bacterial species (33). A second surface protein, SasG, as well as its homologous protein in Staphylococcus epidermidis, Aap, also mediates intercellular interactions and biofilm development in the absence of the ica operon (7, 51). More recently, two independent laboratories have shown that fibronectin binding proteins A and B (FnBPA and FnBPB) induce biofilm development of clinical isolates of S. aureus (45, 55). Finally, there is growing evidence that extracellular DNA, despite not being sufficient to replace PIA/PNAG exopolysaccharide, is an important S. aureus biofilm matrix component (50).During the course of a systematic mutagenesis study of the 17 two-component systems of S. aureus that aimed to identify biofilm-negative regulators, we found that S. aureus agr arlRS double mutants developed an alternative, ica-independent biofilm in a chemically defined medium, Hussain-Hastings-White (HHW) medium (56). This study focused on the identification of the proteinaceous compound responsible for the biofilm developed by S. aureus agr arlRS mutants. Here, we show that S. aureus protein A is responsible for the aggregative phenotype and capacity for biofilm formation displayed by this strain. Furthermore, overproduction of protein A in wild-type S. aureus strains or addition of soluble protein A to bacterial growth medium induced aggregation and biofilm development, suggesting that protein A does not need to be covalently linked to the cell wall to promote multicellular behavior. Moreover, deletion of the spa gene significantly decreased the capacity of S. aureus to colonize subcutaneously implanted catheters. Our findings support a novel role for protein A in promoting multicellular behavior and suggest that protein A-mediated biofilm development may have a critical function during the infection process of S. aureus.     

Two-Component Histidine Phosphotransfer Protein Ypd1 Is Not Essential for Viability in Candida albicans

Prokaryotes and lower eukaryotes, such as yeasts, utilize two-component signal transduction pathways to adapt cells to environmental stress and to regulate the expression of genes associated with virulence. One of the central proteins in this type of signaling mechanism is the phosphohistidine intermediate protein Ypd1. Ypd1 is reported to be essential for viability in the model yeast Saccharomyces cerevisiae. We present data here showing that this is not the case for Candida albicans. Disruption of YPD1 causes cells to flocculate and filament constitutively under conditions that favor growth in yeast form. To determine the function of Ypd1 in the Hog1 mitogen-activated protein kinase (MAPK) pathway, we measured phosphorylation of Hog1 MAPK in ypd1Δ/Δ and wild-type strains of C. albicans. Constitutive phosphorylation of Hog1 was observed in the ypd1Δ/Δ strain compared to the wild-type strain. Furthermore, fluorescence microscopy revealed that green fluorescent protein (GFP)-tagged Ypd1 is localized to both the nucleus and the cytoplasm. The subcellular segregation of GFP-tagged Ypd1 hints at an important role(s) of Ypd1 in regulation of Ssk1 (cytosolic) and Skn7 (nuclear) response regulator proteins via phosphorylation in C. albicans. Overall, our findings have profound implications for a mechanistic understanding of two-component signaling pathways in C. albicans, and perhaps in other pathogenic fungi.     

ENDOSPERM DEFECTIVE1 Is a Novel Microtubule-Associated Protein Essential for Seed Development in Arabidopsis

Early endosperm development involves a series of rapid nuclear divisions in the absence of cytokinesis; thus, many endosperm mutants reveal genes whose functions are essential for mitosis. This work finds that the endosperm of Arabidopsis thaliana endosperm-defective1 (ede1) mutants never cellularizes, contains a reduced number of enlarged polyploid nuclei, and features an aberrant microtubule cytoskeleton, where the specialized radial microtubule systems and cytokinetic phragmoplasts are absent. Early embryo development is substantially normal, although occasional cytokinesis defects are observed. The EDE1 gene was cloned using a map-based approach and represents the pioneer member of a conserved plant-specific family of genes of previously unknown function. EDE1 is expressed in the endosperm and embryo of developing seeds, and its expression is tightly regulated during cell cycle progression. EDE1 protein accumulates in nuclear caps in premitotic cells, colocalizes along microtubules of the spindle and phragmoplast, and binds microtubules in vitro. We conclude that EDE1 is a novel plant-specific microtubule-associated protein essential for microtubule function during the mitotic and cytokinetic stages that generate the Arabidopsis endosperm and embryo.