Eleonora García Véscovi

Serratia marcescens Is Able to Survive and Proliferate in Autophagic-Like Vacuoles inside Non-Phagocytic Cells

Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell.

Serratia marcescens ShlA Pore-Forming Toxin Is Responsible for Early Induction of Autophagy in Host Cells and Is Transcriptionally Regulated by RcsB

ABSTRACT Serratia marcescens is a Gram-negative bacterium that thrives in a wide variety of ambient niches and interacts with an ample range of hosts. As an opportunistic human pathogen, it has increased its clinical incidence in recent years, being responsible for life-threatening nosocomial infections. S. marcescens produces numerous exoproteins with toxic effects, including the ShlA pore-forming toxin, which has been catalogued as its most potent cytotoxin. However, the regulatory mechanisms that govern ShlA expression, as well as its action toward the host, have remained unclear. We have shown that S. marcescens elicits an autophagic response in host nonphagocytic cells. In this work, we determine that the expression of ShlA is responsible for the autophagic response that is promoted prior to bacterial internalization in epithelial cells. We show that a strain unable to express ShlA is no longer able to induce this autophagic mechanism, while heterologous expression of ShlA/ShlB suffices to confer on noninvasive Escherichia coli the capacity to trigger autophagy. We also demonstrate that shlBA harbors a binding motif for the RcsB regulator in its promoter region. RcsB-dependent control of shlBA constitutes a feed-forward regulatory mechanism that allows interplay with flagellar-biogenesis regulation. At the top of the circuit, activated RcsB downregulates expression of flagella by binding to the flhDC promoter region, preventing FliA-activated transcription of shlBA. Simultaneously, RcsB interaction within the shlBA promoter represses ShlA expression. This circuit offers multiple access points to fine-tune ShlA production. These findings also strengthen the case for an RcsB role in orchestrating the expression of Serratia virulence factors.

Two component systems in the spatial program of bacteria.

Despite being considered a relatively simple form of life, bacteria have revealed a high degree of structural organization, with the spatial destination of their components precisely regulated within the cell. Nevertheless, the primary signals that dictate differential distribution of cellular building blocks and physiological processes remain in most cases largely undisclosed. Signal transduction systems are no exception within this three-dimensional organization and two-component systems (TCS) involved in controlling cell division, differentiation, chemotaxis and virulence show specific and/or dynamic localization, engaging in the spatial program of the bacterial cell.

The PhoP/PhoQ System and Its Role in Serratia marcescens Pathogenesis

Serratia marcescens is able to invade, persist, and multiply inside nonphagocytic cells, residing in nonacidic, nondegradative, autophagosome-like vacuoles. In this work, we have examined the physiological role of the PhoP/PhoQ system and its function in the control of critical virulence phenotypes in S. marcescens. We have demonstrated the involvement of the PhoP/PhoQ system in the adaptation of this bacterium to growth on scarce environmental Mg(2+), at acidic pH, and in the presence of polymyxin B. We have also shown that these environmental conditions constitute signals that activate the PhoP/PhoQ system. We have found that the two S. marcescens mgtE orthologs present a conserved PhoP-binding motif and demonstrated that mgtE1 expression is PhoP dependent, reinforcing the importance of PhoP control in magnesium homeostasis. Finally, we have demonstrated that phoP expression is activated intracellularly and that a phoP mutant strain is defective in survival inside epithelial cells. We have shown that the Serratia PhoP/PhoQ system is involved in prevention of the delivery to degradative/acidic compartments.

Unsaturated long-chain free fatty acids are input signals of the Salmonella enterica PhoP/PhoQ regulatory system

Background: The PhoP/PhoQ system governs crucial Salmonella typhimurium pathogenic traits. Results: A screening of natural compounds showed that long chain fatty acids present in the bacterial growth medium down-regulate the PhoP/PhoQ-dependent regulon. Conclusion: Long chain unsaturated fatty acids specifically inhibit PhoQ autokinase activity. Significance: The novel PhoQ input signal reveals a new scenario for the control of Salmonella virulence, providing a rationale for future antibacterial strategies. The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg2+ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the β-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.

Cytolocalization of the PhoP response regulator in Salmonella enterica: modulation by extracellular Mg2+ and by the SCV environment

The PhoP/PhoQ two‐component system plays an essential role regulating numerous virulence phenotypes in Salmonella enterica. Previous work showed that PhoQ, the sensor protein, switches between the kinase‐ and the phosphatase‐dominant state in response to environmental Mg2+ availability. This switch defines the PhoP phosphorylation status and, as a result, the transcriptional activity of this regulator. In this work, using the FlAsH labelling technique, we examine PhoP cytolocalization in response to extracellular Mg2+ limitation in vitro and to the Salmonella‐containing vacuole (SCV) environment in macrophage cells. We demonstrate that in these PhoP/PhoQ‐inducing environments PhoP displays preferential localization to one cell pole, while being homogeneously distributed in the bacterial cytoplasm in repressing conditions. Polar localization is lost in the absence of PhoQ or when a non‐phosphorylatable PhoPD52A mutant is expressed. However, when PhoP transcriptional activation is achieved in a Mg2+‐ and PhoQ‐independent manner, PhoP regains asymmetric polar localization. In addition, we show that, in the analysed conditions, PhoQ cellular distribution does not parallel PhoP location pattern. These findings reveal that PhoP cellular location is dynamic and conditioned by its environmentally defined transcriptional status, showing a new insight in the PhoP/PhoQ system mechanism.

Phosphorylation of serine residues in endogenous proteins of thylakoids and subthylakoid particles in the dark under nonreducing conditions

Abstract Isolated thylakoids, Photosystem I and Photosystem II particles were phosphorylated with [γ-32P]ATP at high specific radioactivity in the dark under nonreducing conditions and in the light in the absence of electron acceptor. The resulting phosphoproteins were compared by gel electrophoresis and autoradiography. Phosphorylation of thylakoids in the dark and in the light rendered distinct patterns of phosphoproteins. Some of the dark-phosphorylated proteins in thylakoids were diminished or not detected in the light-phosphorylated membranes. Phosphorylation of subthylakoid particles was insensitive to light and most of the phosphoproteins in these membranes were also observed in the dark-phosphorylated thylakoids. Dark phosphorylation rendered mostly phosphoserine in individual proteins of thylakoids, subthylakoid particles and lysine-rich histone phosphorylated by the particles. Conversely, phosphothreonine was prevalent in light-phosphorylated thylakoids. The results are consistent with the presence of a protein serine kinase activity that is distributed homogeneously within the thylakoid regions, is more active in the dark, does not require reducing conditions for activity and phosphorylates a number of endogenous substrates most of which belong to the stroma membranes.

Draft Whole-Genome Sequence of Serratia marcescens Strain RM66262, Isolated from a Patient with a Urinary Tract Infection

ABSTRACT Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches and also constitute emergent nosocomial opportunistic pathogens. Here, we report on the draft genome sequence of S. marcescens strain RM66262, which was isolated from a patient with urinary tract infection in the Bacteriology Service of the Rosario National University, Rosario, Argentina.

A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP–PhoQ Regulatory System

INTRODUCTIONnThe PhoP-PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography.nnnOBJECTIVEnTo develop a TLC bioautography to detect inhibitors of the PhoP-PhoQ regulatory system in complex matrixes.nnnMETHODSnThe TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16u2009h.nnnRESULTSnA bioautographic assay suitable for the localisation of inhibitors of the PhoP-PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP-PhoQ system.nnnCONCLUSIONnA practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP-PhoQ regulatory system from a hydrolysed extract.