Infra-red studies of the surface acidity of oxides and zeolites using adsorbed probe molecular

The use of infrared spectroscopy to probe the surface acidity of oxides and molecular sieves is reviewed. The experimental requirements and the type and nature of probe molecules available are also discussed. Special emphasis is given to the criteria that have to be met to arrive at a characterization of the solid that is useful for its catalytic application

Evolutionary adaptation of the essential tRNA methyltransferase TrmD to the signaling molecule 3,5-cAMP in bacteria.

The nucleotide signaling molecule 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) plays important physiological roles, ranging from carbon catabolite repression in bacteria to mediating the action of hormones in higher eukaryotes, including human. However, it remains unclear whether 3',5'-cAMP is universally present in the Firmicutes group of bacteria. We hypothesized that searching for proteins that bind 3',5'-cAMP might provide new insight into this question. Accordingly, we performed a genome-wide screen, and identified the essential Staphylococcus aureus tRNA m1G37 methyltransferase enzyme TrmD, which is conserved in all three domains of life, as a tight 3',5'-cAMP binding protein. TrmD enzymes are known to use S-adenosyl-L-methionine (AdoMet) as substrate; we shown that 3',5'-cAMP binds competitively with AdoMet to the S. aureus TrmD protein, indicating an overlapping binding site. However, the physiological relevance of this discovery remained unclear, as we were unable to identify a functional adenylate cyclase in S. aureus and only detected 2',3'-cAMP but not 3',5'-cAMP in cellular extracts. Interestingly, TrmD proteins from Escherichia coli and Mycobacterium tuberculosis, organisms known to synthesize 3',5'-cAMP, did not bind this signaling nucleotide. Comparative bioinformatics, mutagenesis and biochemical analyses revealed that the highly conserved Tyr86 residue in E. coli TrmD is essential to discriminate between 3',5'-cAMP and the native substrate AdoMet. Combined with a phylogenetic analysis, these results suggest that amino acids in the substrate binding pocket of TrmD underwent an adaptive evolution to accommodate the emergence of adenylate cyclases and thus the signaling molecule 3',5'-cAMP. Altogether this further indicates that S. aureus does not produce 3',5'-cAMP, which would otherwise competitively inhibit an essential enzyme

Differential localization of LTA synthesis proteins and their interaction with the cell division machinery in Staphylococcus aureus

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria. In Staphylococcus aureus it consists of a polyglycerolphosphate-chain that is retained within the membrane via a glycolipid. Using an immunofluorescence approach, we show here that the LTA polymer is not surface exposed in S. aureus, as it can only be detected after digestion of the peptidoglycan layer. S. aureus mutants lacking LTA are enlarged and show aberrant positioning of septa, suggesting a link between LTA synthesis and the cell division process. Using a bacterial two-hybrid approach, we show that the three key LTA synthesis proteins, YpfP and LtaA, involved in glycolipid production, and LtaS, required for LTA backbone synthesis, interact with one another. All three proteins also interacted with numerous cell division and peptidoglycan synthesis proteins, suggesting the formation of a multi-enzyme complex and providing further evidence for the co-ordination of these processes. When assessed by fluorescence microscopy, YpfP and LtaA fluorescent protein fusions localized to the membrane while the LtaS enzyme accumulated at the cell division site. These data support a model whereby LTA backbone synthesis proceeds in S. aureus at the division site in co-ordination with cell division, while glycolipid synthesis takes place throughout the membrane

Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes

Lipoteichoic acid (LTA) is an important cell wall polymer in Gram-positive bacteria. The enzyme responsible for polyglycerolphosphate LTA synthesis is LtaS, first described in Staphylococcus aureus. Four LtaS orthologues, LtaSBS, YfnI, YqgS and YvgJ, are present in Bacillus subtilis. Using an in vitro enzyme assay, we determined that all four proteins are Mn2+-dependent metal enzymes that use phosphatidylglycerol as a substrate. We show that LtaSBS, YfnI and YqgS can produce polymers, suggesting that these three proteins are bona-fide LTA synthases while YvgJ functions as an LTA primase, as indicated by the accumulation of a GroP-Glc2-DAG glycolipid. Western blot analysis of LTA produced by ltaSBS, yfnI, yqgS and yvgJ single, triple and the quadruple mutant, showed that LTA production was only abolished in the quadruple and the YvgJ-only expressing mutant. B. subtilis strains expressing YfnI in the absence of LtaSBS produced LTA of retarded mobility, presumably caused by an increase in chain length as suggested by a structural analysis of purified LTA. Taken together, the presented results indicate that the mere presence or absence of LTA cannot account for cell division and sporulation defects observed in the absence of individual enzymes and revealed an unexpected enzymatic interdependency of LtaS-type proteins in B. subtilis

The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

The Sbi protein of Staphylococcus aureus comprises two IgG-binding domains similar to those of protein A and a region that triggers the activation of complement C3. Sbi is expressed on the cell surface but its C-terminal domain lacks motifs associated with wall or membrane anchoring of proteins in Gram-positive bacteria. Cell-associated Sbi fractionates with the cytoplasmic membrane and is not solubilized during protoplast formation. S. aureus expressing Sbi truncates of the C-terminal Y domain allowed identification of residues that are required for association of Sbi with the membrane. Recombinant Sbi bound to purified cytoplasmic membrane material in vitro and to purified lipoteichoic acid. This explains how Sbi partitions with the membrane in fractionation experiments yet is partially exposed on the cell surface. An LTA-defective mutant of S. aureus had reduced levels of Sbi in the cytoplasmic membrane

Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes

The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen Listeria monocytogenes is intrinsically resistant towards lysozyme, mainly due to deacetylation and O-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An eslB mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less O-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of eslB phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the eslB mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-N-acetylglucosamine (UDP-GlcNAc) production or distribution

Comportamento Dinâmico Do Gerador De Indução Com Dupla Alimentação Orientado No Fluxo Estatórico

Este artigo analisa o impacto da banda passante dos controladores PI de eixo q e d no comportamento dinâmico do Gerador de Indução com Dupla Alimentação (GIDA) orientado no referencial do fluxo estatórico. Com a conecção direta do estator do gerador a rede elétrica, variações na tensão no Ponto de Conecção Comum (PCC) ou variações da corrente dr i causam oscilações no fluxo estatórico. Essas oscilações podem ser amortecidas através da escolha adequada da banda passante dos controladores PI de eixo q e d. Além disso, este artigo contribui com uma análise e metodologia de projeto que garante a estabilidade e bom desempenho do estimador não linear utilizado para obter a posição do fluxo estatórico. Para validar a análise são mostrados resultados experimentais obtidos a partir de um conversor trifásico com modulação Space Vector totalmente controlado através do DSP TMS320F241

Purine nucleosides interfere with c-di-AMP levels and act as adjuvants to re-sensitize MRSA to β-lactam antibiotics

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA β-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased β-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce β-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on β-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced β-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as β-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level β-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the β-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other β-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for β-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of β-lactams against MRSA and potentially other AMR pathogens

PATHOGEN-SPECIFIC ANTIBODY PROFILES IN PATIENTS WITH SEVERE SYSTEMIC INFECTIONS

Infections are often caused by pathobionts, endogenous bacteria that belong to the microbiota. Trauma and surgical intervention can allow bacteria to overcome host defences, ultimately leading to sepsis if left untreated. One of the main defence strategies of the immune system is the production of highly specific antibodies. In the present proof-of-concept study, plasma antibodies against 9 major pathogens were measured in sepsis patients, as an example of severe systemic infections. The binding of plasma antibodies to bacterial extracellular proteins was quantified using a semi-automated immunoblot assay. Comparison of the pathogen-specific antibody levels before and after infection showed an increase in plasma IgG in 20 out of 37 tested patients. This host-directed approach extended the results of pathogen-oriented microbiological and PCR diagnostics: a specific antibody response to additional bacteria was frequently observed, indicating unrecognised poly-microbial invasion. This might explain some cases of failed, seemingly targeted antibiotic treatment