Increased bile resistance in Salmonella enterica mutants lacking Prc periplasmic protease

Prc is a periplasmic protease involved in processing of penicillin-binding protein 3 (PBP3). Lack of Prc suppressesbile sensitivity in Dam-, Wec-, PhoP-, DamX-, and SeqA- mutants of Salmonella enterica, and increases bile resistance in thewild type. Changes in the activity of penicillin binding proteins PBP3, PBP4, PBP5/6 and PBP7 are detected in a Prc-background, suggesting that peptidoglycan remodeling might contribute to bile resistance. [Int Microbiol 2013; 16(2):87-92]Keywords: Salmonella; bile; Prc protease; peptidoglycan; penicillin-binding protein

Peptidoglycan editing in non-proliferating intracellular Salmonella as source of interference with immune signaling

This work was funded by grants PID2020-112971GB-I00/10.13039/501100011033 (F.G-dP.) and PID2019-104070RB-C21 (S.V.) of the Spanish Ministry of Science and Innovation, VR2018-02823 of the Swedish Research Council (F.C.), KAW2012.0184 of the Knut and Alice Wallenberg Foundation (F.C.), and SMK2062 of the Kempe Foundation (F.C.

Peptidoglycan editing in non-proliferating intracellular Salmonella as source of interference with immune signaling

Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan puri- fied from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropep- tides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)- meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This pepti- doglycan has a reduced glycan chain average length and ~30% increase in the L,D-cross- link, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D- transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L, D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhi- murium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remark- ably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-con- taining muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D- alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells.This work was funded by grants PID2020-112971GB-I00/10.13039/501100011033 (F.G-dP.) and PID2019-104070RB-C21 (S.V.) of the Spanish Ministry of Science and Innovation, VR2018-02823 of the Swedish Research Council (F.C.), KAW2012.0184 of the Knut and Alice Wallenberg Foundation (F.C.), and SMK2062 of the Kempe Foundation (F.C.). S.C. was recipient of an EMBO Short-Term Fellowship number 6426 for a stay in the lab of F.C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptPeer reviewe

Life history traits influence in gonad composition of two sympatric species of flatfish

AbstractParalichthys orbignyanus and Paralichthys patagonicus are flatfish with different life history traits, having in common the condition of breeding in seawater. Paralichthys patagonicus remain their whole life in open seawater and Paralichthys orbignyanus are sometimes found in brackish water bodies. As marine and estuarine food webs have different fatty acid (FA) compositions, the aim of this study was to characterize the gonadal maturation of P. orbignyanus and P. patagonicus females through the analysis of lipid content and FA profile in order to understand to what extent life history traits are reflected in the ovarian composition. During gonadal maturation lipid content increased and FA profiles changed in both species, but the lipid increase was greater in P. orbignyanus. The N-3FA and n-3HUFA proportions increased in both species but were higher in P. orbignyanus. The differences between the lifestyles of these species were reflected in the ovarian FA profile mainly as a result of differences in their FA metabolism, causing a greater accumulation of n-3FA and n-3HUFA in P. orbignyanus than in P. patagonicus. The higher lipid accumulation in P. orbignyanus’ ovaries could indicate that this species, feeding in brackish water bodies, has the possibility of storing more energy than P. patagonicus

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p < 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics

Metabolismo del peptidoglicano de Salmonella en el interior de células ecuariotas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-10-2015El peptidoglicano es un componente esencial de la pared celular que rodea por completo a la bacteria y de cuya integridad depende la viabilidad celular. Esta estructura es un heteropolímero formado por largas cadenas glucídicas entrecruzadas por cadenas peptídicas cortas. El peptidoglicano experimenta continuos procesos de remodelado que facilitan la adaptación de la bacteria a condiciones ambientales cambiantes. En estrecha correlación con los diversos enlaces existentes en el peptidoglicano, hoy se conocen una gran cantidad de enzimas implicadas en su metabolismo, a las que hemos dividido, en este trabajo, en cuatro grupos según su actividad: biosíntesis (BIO), modificación (MOD), hidrólisis (HID) y reciclaje (REC). Salmonella enterica es un patógeno intracelular que causa infecciones en un amplio rango de hospedadores. En esta Tesis hemos empleado fibroblastos como modelo de línea celular. En este tipo celular S. enterica serovar Typhimurium (S. Typhimurium) establece un estado de baja proliferación, similar al observado in vivo en bacteria que coloniza tejidos animales. Hemos identificado un total de cuarenta y ocho genes de S. Typhimurium cuyos productos podrían tener una actividad enzimática definida sobre el metabolismo del peptidoglicano. Dentro de estos genes, cinco de ellos, STM1836, STM1910, STM1940, STM3277 y STM4217, se incluyeron en el estudio por estar presentes de forma diferencial en el género Salmonella. STM1836 y STM1910 muestran una identidad del 65 % con los genes mrdA y ftsI, los cuales codifican las proteínas PBP 2 y PBP.3, esenciales en la biosíntesis del peptidoglicano. Por otro lado, STM1940 codifica una enzima con actividad DL-endopeptidasa. La producción de STM1940 aumenta en bacteria intracelular obtenida de células eucariotas, es regulada por el sistema de dos componentes PhoPPhoQ y es necesaria para la virulencia en un modelo de ratón. Durante la infección de fibroblastos se han observado cambios en la maquinaria enzimática del peptidoglicano. Dentro de la célula infectada, S. Typhimurium aumenta la producción de YbiS e YnhG dentro del grupo de enzimas de biosíntesis; PBP 5, PBP 6, PBP 6B, PBP 7, Spr, NlpC y STM1940 como enzimas de modificación; MltA y EmtA dentro de las enzimas de hidrólisis; y NagZ, LdcA y Mpl como enzimas de reciclaje. La existencia de enzimas que reconocen un mismo enlace en el peptidoglicano ha dificultado la obtención de fenotipos en el modelo de infección de fibroblastos. Se ha analizado además la estructura del peptidoglicano de S. Typhimurium obtenido de bacteria intracelular en estado no proliferativo. Este estudio concluyó con la identificación de un componente del peptidoglicano no descrito con anterioridad correspondiente a un muropéptido no entrecruzado que contiene, en la cuarta posición de la cadena peptídica lateral, una molécula de aminoalcohol (alaninol o 1-amino-2-propanol). Esta Tesis describe, de forma completa, el conjunto de enzimas implicadas en el metabolismo del peptidoglicano de S. Typhimurium, centrándose en su regulación en el ambiente extracelular e intracelular además de su contribución a la infección. Información adicional aportada en este trabajo incluye la definición de la estructura del peptidoglicano en bacteria intracelular obtenida de fibroblastos, además del estudio y actividad de la enzima STM1940, codificada en una isla genómica exclusiva del género SalmonellaPeptidoglycan is an essential component of the cell wall that surrounds the bacteria completely and whose integrity ensures bacterial viability against internal osmotic pressure. This structure is a heteropolymer formed by long glycan chains cross-linked by short stem peptide chains. The peptidoglycan is continuously remodeled what facilitates bacterial adaptation to changing environmental conditions. In close relationship to the diversity of bonds known to be present in the peptidoglycan, a large number of enzymes are also involved in its metabolism, which we divided in four groups in this work: biosynthesis (BIO), modification (MOD), hydrolysis (HID) and recycling (REC). Salmonella enterica is an intracellular pathogen that causes infections in a broad range of hosts. In this Thesis, we used fibroblasts as cell line model, since S. enterica serovar Typhimurium (S. Typhimurium) adapts in this cell type to a non-proliferative stage, similar to that observed when bacteria colonize host tissues in vivo. We have identified forty eight genes of S. Typhimurium whose products are predicted to have a well-defined enzymatic activity on the peptidoglycan metabolism. Five of these genes, STM1836, STM1910, STM1940, STM3277 and STM4217, were included in this investigation because they are present almost exclusively in the Salmonella genus. STM1836 and STM1910 exhibit 65 % identity at the amino acid level with the products of mrdA and ftsI, genes that encode PBP 2 and PBP 3, essential biosynthetic proteins involved in the elongation and division phases, respectively. On the other hand, STM1940 encodes an enzyme with DL-endopeptidase activity. The production of STM1940 increase in intracellular bacteria obtained from eukaryotic cells, it is regulated by the two component system PhoP-PhoQ and it is necessary for virulence in the mouse model. During fibroblast infection we have observed changes in the enzymatic machinery of the peptidoglycan. Inside the infected cell, S. Typhimurium increases the production of: the biosynthetic enzymes YbiS and YnhG; the modification enzymes PBP 5, PBP 6, PBP 6B, PBP 7, Spr, NlpC and STM1940; MltA and EmtA as hydrolytic enzymes; and, NagZ, LdcA and Mpl as recycling enzymes. The redundancy of enzymes acting over the same bond of the peptidoglycan has complicated the identification of phenotypes in the fibroblast infection model. We have also analyzed the structure of the peptidoglycan of S. Typhimurium obtained from intracellular bacteria in a non-proliferative state. This study led to the identification of a new component of its peptidoglycan that corresponds to a non-cross-linked muropeptide that contains an aminoalcohol molecule (alaninol or 1-amino-2-propanol) in the fourth position of the peptide chain. Therefore, this Thesis describes in a comprehensive manner, the enzymatic machinery involved in the peptidoglycan metabolism of S. Typhimurium. This study was focused on aspects related to peptidoglycan regulation in bacteria adapting to extracellular and intracellular environments as well as its contribution to the infection. This work also includes the definition of the peptidoglycan structure of non-growing bacteria inside fibroblasts and, in addition, the characterization of a novel DL-endopeptidase, STM1940, encoded in a genomic island exclusive of the Salmonella genu

Increased bile resistance in Salmonella enterica mutants lacking Prc periplasmic protease

Prc is a periplasmic protease involved in processing of penicillin-binding protein 3 (PBP3). Lack of Prc suppresses bile sensitivity in Dam-, Wec-, PhoP-, DamX-, and SeqA- mutants of Salmonella enterica, and increases bile resistance in the wild type. Changes in the activity of penicillin binding proteins PBP3, PBP4, PBP5/6 and PBP7 are detected in a Prc- background, suggesting that peptidoglycan remodeling might contribute to bile resistance.Spanish Government; European Regional Fund (grants BIO2010-15023, BIO2010-18885, and BFU2009-09200); Consejería de Innovación, Ciencia y Empresa; Junta de Andalucía (grant CVI-5879); Spanish Ministry of Education and CulturePeer Reviewe

A novel antisense RNA from the Salmonella virulence plasmid pSLT expressed by non-growing bacteria inside eukaryotic cells.

Bacterial small RNAs (sRNAs) are regulatory molecules playing relevant roles in response to environmental changes, stressful conditions and pathogenesis. The intracellular bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is known to regulate expression of some sRNAs during colonization of fibroblasts. Here, we characterize a previously unknown sRNA encoded in the S. Typhimurium pSLT virulence plasmid that is specifically up-regulated by non-growing dormant bacteria persisting inside fibroblasts. This sRNA was inferred in microarray expression analyses, which unraveled enhanced transcriptional activity in the PSLT047- PSLT046 (mig5) intergenic region. The sRNA transcript was further identified as a 597-nucleotide molecule, which we named IesR-1, for 'Intracellular-expressed-sRNA-1'. IesR-1 expression is low in bacteria growing in axenic cultures across a variety of experimental conditions but displays a marked increase (∼200-300 fold) following bacterial entry into fibroblasts. Remarkably, induction of IesR-1 expression is not prominent in bacteria proliferating within epithelial cells. IesR-1 deletion affects the control of bacterial growth in defined fibroblast cell lines and impairs virulence in a mouse infection model. Expression analyses performed in the PSLT047-iesR-1-PSLT046 (mig5) region support a cis-acting regulatory mechanism of IesR-1 as antisense RNA over the PSLT047 transcript involving interaction at their respective 3' ends and modulation of PSLT047 protein levels. This model is sustained by the scarce production of PSLT047 protein observed in non-growing intracellular bacteria and the high amount of PSLT047 protein produced by bacteria carrying a truncated IesR-1 version with separated 5' and 3' regions. Taken together, these data reveal that S. Typhimurium sRNAs encoded in the pSLT virulence plasmid respond to a state of persistence inside the host cell. As exemplified by IesR-1, some of these sRNAs may contribute to diminish the relative levels of proteins, such as PSLT047, which are probably dispensable for the intracellular lifestyle

A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence

Bacteria remodel peptidoglycan structure in response to environmental changes. Many enzymes are involved in peptidoglycan metabolism; however, little is known about their responsiveness in a defined environment or the modes they assist bacteria to adapt to new niches. Here, we focused in peptidoglycan enzymes that intracellular bacterial pathogens use inside eukaryotic cells. We identified a peptidoglycan enzyme induced by Salmonella enterica serovar Typhimurium in fibroblasts and epithelial cells. This enzyme, which shows γ-D-glutamyl-meso-diaminopimelic acid D,L-endopeptidase activity, is also produced by the pathogen in media with limited nutrients and in resting conditions. The enzyme, termed EcgA for endopeptidase responding to cessation of growth', is encoded in a S. Typhimurium genomic island absent in Escherichia coli. EcgA production is strictly dependent on the virulence regulator PhoP in extra- and intracellular environments. Consistent to this regulation, a mutant lacking EcgA is attenuated in the mouse typhoid model. These findings suggest that specialised peptidoglycan enzymes, such as EcgA, might facilitate Salmonella adaptation to the intracellular lifestyle. Moreover, they indicate that readjustment of peptidoglycan metabolism inside the eukaryotic cell is essential for host colonisation. Many enzymes direct peptidoglycan metabolism but little it is known about their regulation.Fil: Rico-Pérez, Gadea. Consejo Superior de Investigaciones Científicas; EspañaFil: Pezza, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Pucciarelli, M. Graciela. Consejo Superior de Investigaciones Científicas; EspañaFil: de Pedro, Miguel A.. Consejo Superior de Investigaciones Científicas; EspañaFil: Soncini, Fernando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: García del Portillo, Francisco. Consejo Superior de Investigaciones Científicas; Españ

IesR-1 affects the capacity of <i>S.</i> Typhimurium to invade and control growth within the human fibroblast cell line BJ-5ta.

<p><i>S.</i> Typhimurium wild type (white bars) and its isogenic Δ<i>iesR-1</i>/5′ mutant (black bars) were used to infect rat and human fibroblasts (NRK-49F and BJ-5ta, respectively). HeLa epithelial cells were also infected for comparison. Viable intracellular bacteria were counted at 2 h, 6 h (HeLa cells) and 2 h, 24 h (fibroblasts) post-infection. (A) Invasion rates. Bars represent the percentage of bacteria from the initial inoculum that was internalized by the cells upon 30 min of incubation. (B) Intracellular proliferation rates. Bars represent the ratio between the number of viable intracellular bacteria counted at 24 h (fibroblasts) or 6 h (HeLa cells) relative to that determined at 2 h post-infection. Values are the mean ± standard deviation from three independent experiments. (*) <i>P</i><0.05 in student’s t test.</p