Increased Proteolytic Activity of Serratia marcescens Clinical Isolate HU1848 Is Associated with Higher eepR Expression.

Serratia marcescens is a global opportunistic pathogen. In vitro cytotoxicity of this bacterium is mainly related to metalloprotease serralysin (PrtS) activity. Proteolytic capability varies among the different isolates. Here, we characterized protease production and transcriptional regulators at 37°C of two S. marcescens isolates from bronchial expectorations, HU1848 and SmUNAM836. As a reference strain the insect pathogen S. marcescens Db10 was included. Zymography of supernatant cultures revealed a single (SmUNAM836) or double proteolytic zones (HU1848 and Db10). Mass spectrometry confirmed the identity of PrtS and the serralysin-like protease SlpB from supernatant samples. Elevated proteolytic activity and prtS expression were evidenced in the HU1848 strain through azocasein degradation and qRT-PCR, respectively. Evaluation of transcriptional regulators revealed higher eepR expression in HU1848, whereas cpxR and hexS transcriptional levels were similar between studied strains. Higher eepR expression in HU1848 was further confirmed through an in vivo transcriptional assay. Moreover, two putative CpxR binding motifs were identified within the eepR regulatory region. EMSA validated the interaction of CpxR with both motifs. The evaluation of eepR transcription in a cpxR deletion strain indicated that CpxR negatively regulates eepR. Sequence conservation suggests that regulation of eepR by CpxR is common along S. marcescens species. Overall, our data incorporates CpxR to the complex regulatory mechanisms governing eepR expression and associates the increased proteolytic activity of the HU1848 strain with higher eepR transcription. Based on the global impact of EepR in secondary metabolites production, our work contributes to understanding virulence factors variances across S. marcescens isolates.

Dissemination of Gram-positive bacteria to the lung of newborn mice increases local IL-6 and TNFα levels in lethal bacteremia.

Neonatal bacteremia remains the major cause of infectious diseases-related death, especially in preterm newborns. Gram-positive bacteria are the main causative agent of neonatal bacteremia and exhibit a high risk of causing pneumonia and/or meningitis. The pathogenesis of bacteremia in preterm newborn is poorly understood. Current neonatal models of bacterial infection have been used to study the disease mechanisms; however, these studies employed mice of several days of age that could be less comparable to the bacteremia in preterm infants. In this study, we infected intravenously 0-day-old BALB/c mice with different inocula of Staphylococcus aureus, Streptococcus agalactiae or Enterococcus faecalis. We found that the mortality of the newborn mice was inoculum-dependent and also bacterial species-dependent. We observed bacterial burden in the lung, liver, brain, kidney and spleen of the infected animals. The lung was the tissue with the greatest bacterial burden and cellular infiltration in animals infected with the three bacteria evaluated. We found increased production of IL-6 and TNFα in the lung from newborn mice at 3 days post-infection. This neonatal model shows bacterial dissemination to the lung and will be useful for promote a better understanding of the pathophysiology of neonatal pneumonia.

Stimulating the Involvement of Family Members in the Medication Management Activities of Older Adults Through Ambient Displays: Qualitative Study.

Little attention has been paid to how medication management technologies, designed for older adults, modify the participation of family caregivers. We developed a tablet-based ambient display that provides external cues to remind and motivate older adults to take their medications. This study aimed to understand the effect of ambient displays on the involvement of family members in the elderly's medication management. We conducted a 10-week study consisting of interviews administered weekly to nine elderly-caregivers. We identify that new involvement patterns of the family caregivers were provoked through external cues, which made them aware of older adults' medication adherence and encouraged younger relatives to help older adults.

Comparative Analysis of Virulence Profiles of Serratia marcescens Isolated from Diverse Clinical Origins in Mexican Patients.

Background: Serratia marcescens is an enteric bacterium with increasing incidence in clinical settings, attributed mainly to the opportune expression of diverse virulence determinants plus a wide intrinsic and acquired antibiotic resistance. Methods: The aim of this study was to compare the virulence factor profiles of 185 Serratia marcescens isolates from different clinical origins. In vitro proteolytic and hemolytic activities, biofilm formation, and motility were assessed in each strain. Additionally, the pathogenicity of four hypervirulent strains was analyzed in vivo in Galleria mellonella. Results: We found that bacterial isolates from wound/abscess and respiratory tract specimens exhibited the highest protease activity along with a strong biofilm production, while uropathogenic isolates showed the highest hemolytic activity. Swarming and swimming motilities were similar among all the strains. However, respiratory tract isolates showed the most efficient motility. Two hyperhemolytic and two hyperproteolytic strains were detected; the latter were more efficient killing Galleria mellonella with a 50%-60% larval mortality 48 hours after challenge. Conclusion: A correlation was found between biofilm formation and proteolytic and hemolytic activities in biopsy specimens and bloodstream isolates, respectively. Overall, it becomes critical to evaluate and compare the clinical strains virulence diversity in order to understand the underlying mechanisms that allow the establishment and persistence of opportunistic bacterial infections in the host.

Tyrosine Phosphorylation and Dephosphorylation in Burkholderia cenocepacia Affect Biofilm Formation, Growth under Nutritional Deprivation, and Pathogenicity.

Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important posttranslational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF and the low-molecular-weight protein tyrosine phosphatases BCAM0208, BceD, and BCAL2200 of B. cenocepacia K56-2. We show that BCAM1331, BceF, BCAM0208, and BceD contribute to biofilm formation, while BCAL2200 is required for growth under nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low-molecular-weight protein tyrosine phosphatase genes resulted in the attenuation of B. cenocepacia intramacrophage survival and reduced pathogenicity in the Galleria mellonella larval infection model. Experimental evidence indicates that BCAM1331 displays reduced tyrosine autophosphorylation activity compared to that of BceF. With the artificial substrate p-nitrophenyl phosphate, the phosphatase activities of the three low-molecular-weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 became tyrosine phosphorylated in vivo and catalyzed its autodephosphorylation. Together, our data suggest that despite having similar biochemical activities, low-molecular-weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of B. cenocepacia.

ArnT proteins that catalyze the glycosylation of lipopolysaccharide share common features with bacterial N-oligosaccharyltransferases.

ArnT is a glycosyltransferase that catalyzes the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipid A moiety of the lipopolysaccharide. This is a critical modification enabling bacteria to resist killing by antimicrobial peptides. ArnT is an integral inner membrane protein consisting of 13 predicted transmembrane helices and a large periplasmic C-terminal domain. We report here the identification of a functional motif with a canonical consensus sequence DEXRYAX(5)MX(3)GXWX(9)YFEKPX(4)W spanning the first periplasmic loop, which is highly conserved in all ArnT proteins examined. Site-directed mutagenesis demonstrated the contribution of this motif in ArnT function, suggesting that these proteins have a common mechanism. We also demonstrate that the Burkholderia cenocepacia and Salmonella enterica serovar Typhimurium ArnT C-terminal domain is required for polymyxin B resistance in vivo. Deletion of the C-terminal domain in B. cenocepacia ArnT resulted in a protein with significantly reduced in vitro binding to a lipid A fluorescent substrate and unable to catalyze lipid A modification with l-Ara4N. An in silico predicted structural model of ArnT strongly resembled the tertiary structure of Campylobacter lari PglB, a bacterial oligosaccharyltransferase involved in protein N-glycosylation. Therefore, distantly related oligosaccharyltransferases from ArnT and PglB families operating on lipid and polypeptide substrates, respectively, share unexpected structural similarity that could not be predicted from direct amino acid sequence comparisons. We propose that lipid A and protein glycosylation enzymes share a conserved catalytic mechanism despite their evolutionary divergence.

EscO, a functional and structural analog of the flagellar FliJ protein, is a positive regulator of EscN ATPase activity of the enteropathogenic Escherichia coli injectisome.

Type III secretion systems (T3SSs) are multiprotein molecular devices used by many Gram-negative bacterial pathogens to translocate effector proteins into eukaryotic cells. A T3SS is also used for protein export in flagellar assembly, which promotes bacterial motility. The two systems are evolutionarily related, possessing highly conserved components in their export apparatuses. Enteropathogenic Escherichia coli (EPEC) employs a T3SS, encoded by genes in the locus of enterocyte effacement (LEE) pathogenicity island, to colonize the human intestine and cause diarrheal disease. In the present work, we investigated the role of the LEE-encoded EscO protein (previously Orf15 or EscA) in T3SS biogenesis. We show that EscO shares similar properties with the flagellar FliJ and the Yersinia YscO protein families. Our findings demonstrate that EscO is essential for secretion of all categories of T3SS substrates. Consistent with its central role in protein secretion, it was found to interact with the ATPase EscN and its negative regulator, EscL, of the export apparatus. Moreover, we show that EscO stimulates EscN enzymatic activity; however, it is unable to upregulate ATP hydrolysis in the presence of EscL. Remarkably, EscO partially restored the swimming defect of a Salmonella flagellar fliJ mutant and was able to stimulate the ATPase activity of FliI. Overall, our data indicate that EscO is the virulence counterpart of the flagellar FliJ protein.

A Burkholderia cenocepacia gene encoding a non-functional tyrosine phosphatase is required for the delayed maturation of the bacteria-containing vacuoles in macrophages.

Burkholderia cenocepacia infects patients with cystic fibrosis. We have previously shown that B. cenocepacia can survive in macrophages within membrane vacuoles [B. cenocepacia-containing vacuoles (BcCVs)] that preclude fusion with the lysosome. The bacterial factors involved in B. cenocepacia intracellular survival are not fully elucidated. We report here that deletion of BCAM0628, encoding a predicted low molecular weight protein tyrosine phosphatase (LMW-PTP) that is restricted to B. cenocepacia strains of the transmissible ET-12 clone, accelerates the maturation of the BcCVs. Compared to the parental strain and deletion mutants in other LMW-PTPs that are widely conserved in Burkholderia species, a greater proportion of BcCVs containing the ΔBCAM0628 mutant were targeted to the lysosome. Accelerated BcCV maturation was not due to reduced intracellular viability since ΔBCAM0628 survived and replicated in macrophages similarly to the parental strain. Therefore, BCAM0628 was referred to as dpm (delayed phagosome maturation). We provide evidence that the Dpm protein is secreted during growth in vitro and upon macrophage infection. Dpm secretion requires an N-terminal signal peptide. Heterologous expression of Dpm in Burkholderia multivorans confers to this bacterium a similar phagosomal maturation delay to that found with B. cenocepacia. We demonstrate that Dpm is an inactive phosphatase, suggesting that its contribution to phagosomal maturation arrest must be unrelated to tyrosine phosphatase activity.

Toll-like receptor 4 gene polymorphisms and acute coronary syndrome: No association in a Mexican population / No existe asociación entre los polimorfismos del receptor tipo toll-4 y el síndrome coronario agudo

Objective: The objective of this study was to establish the role of the TLR-4 gene polymorphisms in individuals in risk of developing ACS. Methods: The study included 457 Mexican patients with ACS and 283 control individuals. The TLR-4 Asp299Gly and TLR-4 Thr399Ile single nucleotide polymorphisms were genotyped using 5' exonuclease TaqMan genotyping assays on an 7900HT Fast real-time PCR system according to manufacturer's instructions (Applied Biosystems, Foster City, USA). Results: The results obtained in this study showed that the frequency of the two polymorphisms (TLR-4 Asp299Gly and TLR-4 Thr399Ile) studied were similar between patients with ACS and healthy controls. Multiple logistic regression analysis showed that the largest risk factor for ACS development was given by smoking (11.88-fold increased risk), hypertension (4.32-fold increased risk), type II diabetes (3.44-fold increased risk), gender (2.32-fold increased risk), and dyslipidemia (1.52-fold increased risk). Conclusion: The Asp299Gly and Thr399Ile polymorphisms were not associated with susceptibility to ACS in the Mexican population.

Objetivo: El objetivo de este estudio fue establecer el papel de los polimorfismos del gen TLR-4 en individuos en riesgo de desarrollar síndrome coronario agudo (SCA). Métodos: El estudio incluyó a 457 pacientes mexicanos con SCA y 283 individuos como grupo control. Los polimorfismos de un solo nucleótido TLR-4 Asp299Gly y TLR-4 Thr399Ile fueron determinados usando ensayos TaqMan 5' exonucleasa en un equipo tiempo real 7900HT (Fast real-time PCR system) de acuerdo con instrucciones del fabricante Applied Biosystems, Foster City, EE.UU. Resultados: Los resultados obtenidos mostraron que las frecuencias de los 2 polimorfismos (TLR-4Asp299Gly y TLR-4 Thr399Ile) estudiados fueron similares entre pacientes con SCA e individuos control. No obstante, el análisis de regresión logística mostró que los factores de mayor riesgo para desarrollar SCA se debieron a tabaquismo (incrementa 11.88 veces el riesgo), hipertensión (incrementa 4.32 veces el riesgo), diabetes tipo 2 (incrementa 3.44 veces el riesgo), género (incrementa 2.32 veces el riesgo), y la dislipidemia (incrementa 1.52 veces el riesgo). Conclusión: Determinamos que los polimorfismos Asp299Gly y Thr399Ile no se asocian con la susceptibilidad al SCA en la población mexicana.

Toll-like receptor 4 gene polymorphisms and acute coronary syndrome: no association in a Mexican population.

OBJECTIVE: The objective of this study was to establish the role of the TLR-4 gene polymorphisms in individuals in risk of developing ACS. METHODS: The study included 457 Mexican patients with ACS and 283 control individuals. The TLR-4 Asp299Gly and TLR-4 Thr399Ile single nucleotide polymorphisms were genotyped using 5' exonuclease TaqMan genotyping assays on an 7900HT Fast real-time PCR system according to manufacturer's instructions (Applied Biosystems, Foster City, USA). RESULTS: The results obtained in this study showed that the frequency of the two polymorphisms (TLR-4 Asp299Gly and TLR-4 Thr399Ile) studied were similar between patients with ACS and healthy controls. Multiple logistic regression analysis showed that the largest risk factor for ACS development was given by smoking (11.88-fold increased risk), hypertension (4.32-fold increased risk), type II diabetes (3.44-fold increased risk), gender (2.32-fold increased risk), and dyslipidemia (1.52-fold increased risk). CONCLUSION: The Asp299Gly and Thr399Ile polymorphisms were not associated with susceptibility to ACS in the Mexican population.

Enzymatic characterization of the enteropathogenic Escherichia coli type III secretion ATPase EscN.

Type III secretion is a transport mechanism by which bacteria secrete proteins across their cell envelope. This protein export pathway is used by two different bacterial nanomachines: the flagellum and the injectisome. An indispensable component of these secretion systems is an ATPase similar to the F1-ATPase beta subunit. Here we characterize EscN, an enteropathogenic Escherichia coli type III ATPase. A recombinant version of EscN, which was fully functional in complementation tests, was purified to homogeneity. Our results demonstrate that EscN is a Mg2+-dependent ATPase (kcat 0.35 s(-1)). We also define optimal conditions for the hydrolysis reaction. EscN displays protein concentration-dependent activity, suggesting that the specific activity changes with the oligomeric state of the protein. The presence of active oligomers was revealed by size exclusion chromatography and native gel electrophoresis.