Characterization of the outer membrane subproteome of the virulent strain Salmonella Typhimurium SL1344.

Outer membrane proteins (OMPs) play an important role in the interaction of bacterial pathogens with host cells. Indeed, some OMPs from different Gram-negative bacteria have been recognized as important virulence factors for host immune recognition. This scenario has led to the study of the outer membrane (OM) subproteome of pathogenic bacteria as an essential step for gaining insight into the mechanisms of pathogenesis and for the identification of virulence factors. Although progress in the characterization of the OM has recently been reported, detailed protein composition of this subcellular localization has not been clearly defined for most pathogens. Salmonella enterica serovar Typhimurium is not only a leading cause of human gastroenteritis in high-income countries but is also one of the main causes of invasive non-typhoidal salmonellosis (iNTS) in middle- and low-income countries. The incidence of non-typhoidal salmonellosis is increasing worldwide, causing millions of infections and deaths among humans each year. Regrettably, antimicrobial resistance to a broad spectrum of antibiotics is common among non-Typhi Salmonella strains. Therefore, the development of vaccines targeting this leading invasive pathogen is warranted. In the present study we have identified the OM protein profile of the virulent S. Typhimurium strain SL1344 by means of sarkosyl extraction. SIGNIFICANCE: Salmonella enterica serovar Typhimurium causes food-borne gastroenteritis around the world, but is also responsible for a more serious manifestation of the disease through a form of invasive illness, invasive non-typhoidal Salmonella (iNTS) disease, which is considered a major public health problem in low- and middle-income countries. Even though some studies have been carried out in order to characterize the outer membrane subproteome of this human pathogen, as far as we know, this is the first report in which the most indicated methodology has been used in order to extract the outer membrane proteins and to check the presence of the proteins in the SL1344 genome; indeed all the previous studies were carried out before the genome sequence was available in 2012. Outer membrane proteins are key elements for the interaction of Gram-negative bacteria with their environment ­ including the host ­ and have fundamental roles in both infection and resistance processes. Therefore, a detailed knowledge of the outer membrane composition will certainly play a key role in providing new targets to fight this pathogen in further studies.

Automated categorization of methicillin-resistant Staphylococcus aureus clinical isolates into different clonal complexes by MALDI-TOF mass spectrometry.

Early identification of methicillin-resistant Staphylococcus aureus (MRSA) dominant clones involved in infection and initiation of adequate infection control measures are essential to limit MRSA spread and understand MRSA population dynamics. In this study we evaluated the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) for the automated discrimination of the major MRSA lineages (clonal complexes, CC) identified in our hospital during a 20-year period (1990-2009). A collection of 82 well-characterized MRSA isolates belonging to the four main CCs (CC5, CC8, CC22 and CC398) was split into a reference set (n = 36) and a validation set (n = 46) to generate pattern recognition models using the ClinProTools software for the identification of MALDI-TOF/MS biomarker peaks. The supervised neural network (SNN) model showed the best performance compared with two other models, with sensitivity and specificity values of 100% and 99.11%, respectively. Eleven peaks (m/z range: 3278-6592) with the highest separation power were identified and used to differentiate all four CCs. Validation of the SNN model using ClinProTools resulted in a positive predictive value (PPV) of 99.6%. The specific contribution of each peak to the model was used to generate subtyping reference signatures for automated subtyping using the BioTyper software, which successfully classified MRSA isolates into their corresponding CCs with a PPV of 98.9%. In conclusion, we find this novel automated MALDI-TOF/MS approach to be a promising, powerful and reliable tool for S. aureus typing.

Implementation of MALDI-TOF MS technology for the identification of clinical isolates of Mycobacterium spp. in mycobacterial diagnosis.

A total of 243 clinical isolates of the Mycobacterium genus were studied, 143 and 100 using two protocols (Protocol v2 and Protocol v3, respectively) provided by the manufacturer. The overall correlation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with the standard identification methods was 63.8 %. The rate of misidentification was 3.2 %, mainly affecting very close species. In Protocol v2, the correlation was 57.3 %, being greater in solid than in liquid media (71.7 % vs. 44.7 %, p < 0.05). Albeit not significant, a trend to a greater correlation for M. tuberculosis complex compared to non-tuberculous mycobacteria (NTM) (63.6 % vs. 55.5 %) was observed. In Protocol v3, the correlation was 73 %, with no significant differences between solid and liquid media (70.8 % vs. 75 %). In conclusion, MALDI-TOF MS may play a role in identifying mycobacterial species isolated from clinical samples, being faster than sequencing and hybridization-based techniques.

Mechanism of the family 1 beta-glucosidase from Streptomyces sp: catalytic residues and kinetic studies.

The Streptomyces sp. beta-glucosidase (Bgl3) is a retaining glycosidase that belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-nitrophenyl beta-D-glycosides revealed that the highest k(cat)/K(M) values are obtained with glucoside (with strong substrate inhibition) and fucoside (with no substrate inhibition) substrates and that Bgl3 has 10-fold glucosidase over galactosidase activity. Reactivity studies by means of a Hammett analysis using a series of substituted aryl beta-glucosides gave a biphasic plot log k(cat) vs pK(a) of the phenol aglycon: a linear region with a slope of beta(lg) = -0.8 for the less reactive substrates (pK(a) > 8) and no significant dependence for activated substrates (pK(a) < 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glycosyl-enzyme intermediate is rate limiting for the former substrates, while hydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family 1 beta-glucosidases, glutamic acids 178 and 383 were changed to glutamine and alanine by site-directed mutagenesis. Mutation of Glu178 to Gln and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (10(5)-10(6)-fold) were obtained for mutants at Glu383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exogenous nucleophile. The E178A mutant yielded the beta-glucosyl azide adduct (by (1)H NMR) with a 200-fold increase on k(cat) for the 2,4-dinitrophenyl glucoside but constant k(cat)/K(M) on azide concentration. On the other hand, the E383A mutant with the same substrate gave the alpha-glucosyl azide product and a 100-fold increase in k(cat) at 1 M azide. In conclusion, Glu178 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bgl3 beta-glucosidase displays kinetic and mechanistic properties similar to other family 1 enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.