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Objective Detecting traces of ancient DNA of Vibrio cholerae to provide genetic information associated with the fifth cholera pandemic. Materials Sediment samples from the sacral foramina of four individuals were analyzed, recovered from a mass grave near an institution dedicated exclusively to the isolation and treatment of citizens infected with cholera in the late 19th century in the city of Cordoba, Argentina. Methods Paleogenetic techniques (ancient DNA extraction, PCR amplification, and Sanger sequencing) were applied. Specific primers for Vibrio cholerae (VCR, ctxA, ctxB, and tcpA) were designed. Results By amplifying and sequencing the Vibrio cholerae repeats fragment, the infection in at least one individual was confirmed. Conclusions The synthesis of the paleogenetic results with the archaeological and historical evidence strongly supports that at least one individual from the mass grave in Cordoba, Argentina, was a victim of the fifth cholera pandemic. Significance Confirming the presence of the disease through multiple lines of evidence, including genetic, archaeological, and historical analyses, strengthens and affirms our understanding of the presence, effects, and potential evolutionary paths of the disease in the past. Limitations Vibrio cholerae repeats were sequenced in one individual, while the remaining genes could not be amplified, which is likely related to gene copy number. Suggestions for future research Paleogenetic examination of ancient samples from different locations will broaden our understanding of the origin, evolution, and past dissemination of Vibrio cholerae epidemic strains.

Enterobacteriaceae are a major cause of bloodstream infections and their antimicrobial resistance continues to increase. This leads to higher morbidity-mortality rates and public health costs. Carbapenem-resistant Enterobacteriaceae represent a serious challenge globally, since there are few therapeutic options available. Aim: Clinical/microbiological characterization of the carbapenem-resistant bacteremia observed over a period of 4 years. Methods: Retrospective, observational and descriptive study about bacteremia caused by carbapenem-resistant and susceptible Enterobacteriaceae. Results: A total of 84 patients with bacteremia including carbapenem-resistant and susceptible Enterobacteriaceae were analyzed. We found that patients infected with carbapenemresistant strains presented a higher proportion of: previous antibiotic treatment, hospitalization in intensive care unit (ICU), onset of the bacteremia during hospitalization in ICU and previous infection with extended-spectrum-beta-lactamase producing Enterobacteriaceae. Additionally, we observed a predominance of KPC-producing Klebsiella pneumoniae and an attributable mortality rate of 52.4%. Discussion: This study allowed for a better understanding of an emerging problem with high mortality, which in turn is useful for the design and adoption of infection control strategies and effective treatment regimens adapted to our local epidemiology. Fil: Lipari, Flavio Gabriel. Hospital Privado Universitario de Córdoba; Argentina Fil: Hernández, Daniela. Hospital Privado Universitario de Córdoba; Argentina Fil: Vilaró, Mario. Hospital Privado Universitario de Córdoba; Argentina Fil: Caeiro, Juan Pablo. Hospital Privado Universitario de Córdoba; Argentina Fil: Saka, Hector Alex. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen in humans and a frequent cause of asymptomatic, persistent infections leading to serious complications, particularly in young women. Chlamydia displays a unique obligate intracellular lifestyle involving the infectious elementary body and the replicative reticulate body. In the presence of stressors such as gamma-interferon (IFNγ) or beta-lactam antibiotics, C. trachomatis undergoes an interruption in its replication cycle and enters a viable but non-cultivable state. Upon removal of the stressors, surviving C. trachomatis resume cell division and developmental transitions. In this report, we describe a genetic screen to identify C. trachomatis mutants with defects in recovery from IFNγ- and/or penicillin-induced stress and characterized a chemically derived C. trachomatis mutant strain that exhibited a significant decrease in recovery from IFNγ- but not penicillin-induced stress. Through lateral gene transfer and targeted insertional gene inactivation we identified ptr, encoding a predicted protease, as a gene required for recovery from IFNγ-induced stress. A C. trachomatis LGV-L2 ptr-null strain displayed reduced generation of infectious progeny and impaired genome replication upon removal of IFNγ. This defect was restored by introducing a wild type copy of ptr on a plasmid, indicating that Ptr is required for a rapid growth upon removal of IFN?. Ptr was expressed throughout the developmental cycle and localized to the inclusion lumen. Overall, our findings indicate that the putative secreted protease Ptr is required for C. trachomatis to specifically recover from IFNγ- but not penicillin-induced stress. Fil: Panzetta, Maria Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Lujan, Agustin Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Bastidas, Robert J.. University of Duke; Estados Unidos Fil: Damiani, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Valdivia, Raphael H.. University of Duke; Estados Unidos Fil: Saka, Hector Alex. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

The Chlamydiaceae comprise a group of highly adapted bacterial pathogens sharing a unique intracellular lifestyle. Three Chlamydia species are pathogenic to humans: Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci. C. trachomatis is the leading bacterial cause of sexually-transmitted infections and infectious blindness worldwide. Chlamydia pneumoniae is a major cause of community-acquired atypical pneumonia. C. psittaci primarily affects psittacine birds and can be transmitted to humans causing psittacosis, a potentially fatal form of pneumonia. As opposed to other bacterial pathogens, the spread of clinically relevant antimicrobial resistance genes does not seem to be a major problem for the treatment of Chlamydia infections. However, when exposed to stressing conditions, like those arising from exposure to antimicrobial stimuli, these bacteria undergo a temporary interruption in their replication cycle and enter a viable but non-cultivable state known as persistence. When the stressing conditions are removed, Chlamydia resumes replication and generation of infectious particles. This review gives an overview of the different survival strategies used by Chlamydia to evade the deleterious effects of penicillin and IFNγ, with a focus on the different models used to study Chlamydia persistence, their contribution to elucidating the molecular basis of this complex phenomenon and their potential implications for studies in animal models of infection. Fil: Panzetta, Maria Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Valdivia, Raphael H.. University of Duke; Estados Unidos Fil: Saka, Hector Alex. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Chlamydia trachomatis is the most commonly reported agent of sexually transmitted bacterial infections worldwide. This pathogenfrequently leads to persistent, long-term, subclinical infections, which in turn may cause severe pathology in susceptible hosts. This isin part due to the strategies that Chlamydia trachomatis uses to survive within epithelial cells and to evade the host immune response,such as subverting intracellular trafficking, interfering signaling pathways and preventing apoptosis. Innate immune receptors such astoll-like receptors expressed on epithelial and immune cells in the genital tract mediate the recognition of chlamydial molecularpatterns. After bacterial recognition, a subset of pro-inflammatory cytokines and chemokines are continuously released by epithelialcells. The innate immune response is followed by the initiation of the adaptive response against Chlamydia trachomatis, which in turnmay result in T helper 1-mediated protection or in T helper 2-mediated immunopathology. Understanding the molecular mechanismsdeveloped by Chlamydia trachomatis to avoid killing and host immune response would be crucial for designing new therapeuticapproaches and developing protective vaccines. In this review, we focus on chlamydial survival strategies and the elicited immuneresponses in male genital tract infections. Fil: Mackern Oberti, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo; Argentina Fil: Motrich, Ruben Dario. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Damiani, María Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Saka, Hector Alex. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Quintero, Cristian Andre. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Sanchez, Leonardo Rodolfo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Moreno Sosa, María Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Cuffini, Cecilia Gabriela. Universidad Nacional de Cordoba. Facultad de Medicina. Instituto de Virología; Argentina Fil: Rivero, Virginia Elena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

The secreted Chlamydia protease CPAF cleaves a defined set of mammalian and Chlamydia proteins in vitro. As a result, this protease has been proposed to modulate a range of bacterial and host cellular functions. However, it has recently come into question the extent to which many of its identified substrates constitute bona fide targets of proteolysis in infected host cell rather than artifacts of postlysis degradation. Here, we clarify the role played by CPAF in cellular models of infection by analyzing Chlamydia trachomatis mutants deficient for CPAF activity. Using reverse genetic approaches, we identified two C. trachomatis strains possessing nonsense, loss of function mutations in cpa (CT858) and a third strain containing a mutation in type II secretion (T2S) machinery that inhibited CPAF activity by blocking zymogen secretion and subsequent proteolytic maturation into the active hydrolase. HeLa cells infected with T2S− or CPAF−C. trachomatis mutants lacked detectable in vitro CPAF proteolytic activity and were not defective for cellular traits that have been previously attributed to CPAF activity, including resistance to staurosporine induced apoptosis, Golgi fragmentation, altered NFκB dependent gene expression, and resistance to reinfection. However, CPAF deficient mutants did display impaired generation of infectious elementary bodies (EBs), indicating an important role for this protease in the full replicative potential of C. trachomatis. In addition, we provide compelling evidence in live cells that CPAF mediated protein processing of at least two host protein targets, vimentin filaments and the nuclear envelope protein lamin associated protein 1 (LAP1), occurs rapidly after the loss of the inclusion membrane integrity, but before loss of plasma membrane permeability and cell lysis. CPAF dependent processing of host proteins correlates with a loss of inclusion membrane integrity, and so we propose that CPAF plays a role late in infection, possibly during the stages leading to the dismantling of the infected cell prior to the release of EBs during cell lysis. Fil: Snavely, Emily. University of Duke; Estados Unidos Fil: Kokes, Marcela. University of Duke; Estados Unidos Fil: Dunn, Joe Dan. University of Duke; Estados Unidos Fil: Saka, Hector Alex. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Duke; Estados Unidos Fil: Nguyen, Bidong D.. University of Duke; Estados Unidos Fil: Bastidas, Robert J.. University of Duke; Estados Unidos Fil: McCafferty, Dewey G.. University of Duke; Estados Unidos Fil: Valdivia, Raphael H.. University of Duke; Estados Unidos

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection. However, whether Chlamydia trachomatis has a negative impact on sperm quality and male fertility is still controversial. Herein, we report the effects on sperm quality of the in vitro exposure of spermatozoa to Chlamydia trachomatis, and also the effects of male genital infection on male fertility using an animal model. Human and mouse sperm were obtained from healthy donors and cauda epididimys from C57BL/6 mice, respectively. Highly motile human or mouse spermatozoa were in vitro exposed to C. trachomatis (serovar E or LGV) or C. muridarum, respectively. Then, sperm quality parameters were analyzed. Moreover, male fertility of Chlamydia muridarum infected male C57BL/6 mice was assessed. Human or murine sperm in vitro exposed to increasing bacterial concentrations or soluble factors from C. trachomatis or C. muridarum, respectively, did not show differences in sperm motility and viability, apoptosis, mitochondrial membrane potential, DNA fragmentation, ROS production and lipid peroxidation levels, when compared with control sperm (p > 0.05). Moreover, no differences in fertility parameters (potency, fecundity, fertility index, pre-and post-implantation loss) were observed between control and infected males. In conclusion, our results indicate that Chlamydia spp. neither directly exerts deleterious effects on spermatozoa nor impairs male fertility. Fil: Puerta Suarez, Jenniffer. Universidad de Antioquia; Colombia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Sanchez, Leonardo Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Salazar, Florencia. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Saka, Hector Alex. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Molina, Rosa Alejandra. Laboratorio de Andrología y Reproducción; Argentina Fil: Tissera, Andrea Daniela. Laboratorio de Andrología y Reproducción; Argentina Fil: Rivero, Virginia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Cardona Maya, Walter D.. Universidad de Antioquia; Colombia Fil: Motrich, Ruben Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba; Argentina

Lipid droplets (LDs) are neutral lipid storage organelles ubiquitous to eukaryotic cells. It is increasingly recognized that LDs interact extensively with other organelles and that they perform functions beyond passive lipid storage and lipid homeostasis. One emerging function for LDs is the coordination of immune responses, as these organelles participate in the generation of prostaglandins and leukotrienes, which are important inflammation mediators. Similarly, LDs are also beginning to be recognized as playing a role in interferon responses and in antigen cross presentation. Not surprisingly, there is emerging evidence that many pathogens, including hepatitis C and Dengue viruses, Chlamydia, and Mycobacterium, target LDs during infection either for nutritional purposes or as part of an anti-immunity strategy. We here review recent findings that link LDs to the regulation and execution of immune responses in the context of host-pathogen interactions.

Chlamydia trachomatis is an obligate intracellular pathogen responsible for ocular and genital infections of significant public health importance. C. trachomatis undergoes a biphasic developmental cycle alternating between two distinct forms: the infectious elementary body (EB), and the replicative but non-infectious reticulate body (RB). The molecular basis for these developmental transitions and the metabolic properties of the EB and RB forms are poorly understood as these bacteria have traditionally been difficult to manipulate through classical genetic approaches. Using two-dimensional liquid chromatography - tandem mass spectrometry (LC/LC-MS/MS) we performed a large-scale, label-free quantitative proteomic analysis of C. trachomatis LGV-L2 EB and RB forms. Additionally, we carried out LC-MS/MS to analyse the membranes of the pathogen-containing vacuole ('inclusion'). We developed a label-free quantification approaches to measure protein abundance in a mixed-proteome background which we applied for EB and RB quantitative analysis. In this manner, we catalogued the relative distribution of > 54% of the predicted proteins in the C. trachomatis LGV-L2 proteome. Proteins required for central metabolism and glucose catabolism were predominant in the EB, whereas proteins associated with protein synthesis, ATP generation and nutrient transport were more abundant in the RB. These findings suggest that the EB is primed for a burst in metabolic activity upon entry, whereas the RB form is geared towards nutrient utilization, a rapid increase in cellular mass, and securing the resources for an impending transition back to the EB form. The most revealing difference between the two forms was the relative deficiency of cytoplasmic factors required for efficient type III secretion (T3S) in the RB stage at 18 h post infection, suggesting a reduced T3S capacity or a low frequency of active T3S apparatus assembled on a 'per organism' basis. Our results show that EB and RB proteomes are streamlined to fulfil their predicted biological functions: maximum infectivity for EBs and replicative capacity for RBs.

The Chlamydiae are obligate intracellular pathogen that replicate within a membrane-bound vacuole, termed the “inclusion”. From this compartment, bacteria acquire essential nutrients by selectively redirecting transport vesicles and hijacking intracellular organelles. Re-routing is achieved by several mechanisms including proteolysis-mediated fragmentation of the Golgi apparatus, recruitment of Rab GTPases and SNAREs, and translocation of cytoplasmic organelles into the inclusion lumen. Given Chlamydiae’s extended co-evolution with eukaryotic cells, it is likely that co-option of multiple cellular pathways is a strategy to provide redundancy in the acquisition of essential nutrients from the host and has contributed to the success of these highly adapted pathogens.

Epidemiological surveillance for community-associated methicillin-resistant Staphylococcus aureus revealed prevalences of 33% and 13% in pediatric and adult patients, respectively, in Cordoba, Argentina, in 2005. This study describes for the first time the emergence and dissemination of the sequence type 5 (ST5) lineage as the most prevalent clone (89%) (pulsed-field gel electrophoresis type I-ST5-staphylococcal cassette chromosome type IVa- spa type 311) harboring the Panton-Valentine leukocidin and enterotoxin A genes.

Autophagy is the unique, regulated mechanism for the degradation of organelles. This intracellular process acts as a prosurvival pathway during cell starvation or stress and is also involved in cellular response against specific bacterial infections. Vibrio cholerae is a noninvasive intestinal pathogen that has been studied extensively as the causative agent of the human disease cholera. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. Besides cholera toxin, this bacterium secretes a hemolytic exotoxin termed V. cholerae cytolysin (VCC) that causes extensive vacuolation in epithelial cells. In this work, we explored the relationship between the vacuolation caused by VCC and the autophagic pathway. Treatment of cells with VCC increased the punctate distribution of LC3, a feature indicative of autophagosome formation. Moreover, VCC-induced vacuoles colocalized with LC3 in several cell lines, including human intestinal Caco-2 cells, indicating the interaction of the large vacuoles with autophagic vesicles. Electron microscopy analysis confirmed that the vacuoles caused by VCC presented hallmarks of autophagosomes. Additionally, biochemical evidence demonstrated the degradative nature of the VCC-generated vacuoles. Interestingly, autophagy inhibition resulted in decreased survival of Caco-2 cells upon VCC intoxication. Also, VCC failed to induce vacuolization in Atg 5−/− cells, and the survival response of these cells against the toxin was dramatically impaired. These results demonstrate that autophagy acts as a cellular defense pathway against secreted bacterial toxins.

In a previous study, an analysis of 77 ampicillin-nonsusceptible (resistant plus intermediate categories) strains of Vibrio cholerae non-O1, non-O139, isolated from aquatic environment and diarrheal stool, showed that all of them produced a β-lactamase with a pI of 5.4. Hybridization or amplification by PCR with a probe for bla TEM or primers for bla CARB gene families was negative. In this work, an environmental ampicillin-resistant strain from this sample, ME11762, isolated from a waterway in the west region of Argentina, was studied. The nucleotide sequence of the structural gene of the β-lactamase was determined by bidirectional sequencing of a Sau 3AI fragment belonging to this isolate. The gene encodes a new 288-amino-acid protein, designated CARB-7, that shares 88.5% homology with the CARB-6 enzyme; an overall 83.2% homology with PSE-4, PSE-1, CARB-3, and the Proteus mirabilis N29 enzymes; and 79% homology with CARB-4 enzyme. The gene for this β-lactamase could not be transferred to Escherichia coli by conjugation. The nucleotide sequence of the flanking regions of the bla CARB-7 gene showed the occurrence of three 123-bp V. cholerae repeated sequences, all of which were found outside the predicted open reading frame. The upstream fragment of the bla CARB-7 gene shared 93% identity with a locus situated inside V. cholerae 's chromosome 2. These results strongly suggest the chromosomal location of the bla CARB-7 gene, making this the first communication of a β-lactamase gene located on the VCR island of the V. cholerae genome.

MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin. Fil: Furuse, Yuki. University of Duke; Estados Unidos Fil: Finethy, Ryan. University of Duke; Estados Unidos Fil: Saka, Hector Alex. University of Duke; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Xet Mull, Ana M.. University of Duke; Estados Unidos Fil: Sisk, Dana M.. University of Duke; Estados Unidos Fil: Jurcic Smith, Kristen L.. University of Duke; Estados Unidos Fil: Lee, Sunhee. University of Duke; Estados Unidos Fil: Coers, Jörn. University of Duke; Estados Unidos Fil: Valdivia, Raphael H.. University of Duke; Estados Unidos Fil: Tobin, David M.. University of Duke; Estados Unidos Fil: Cullen, Bryan R.. University of Duke; Estados Unidos

Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry. We identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form large molecular weight complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to EBs at entry sites where it remained associated with nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia., Author Summary Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes a range of human diseases of significant public health importance. To create a suitable replicative niche within its host, Chlamydia delivers effector proteins across mammalian membranes via a syringe-like apparatus termed a Type III secretion (T3S) system. The lack of a robust system for the molecular genetic manipulation of these pathogens has hindered progress in identifying and characterizing T3S effectors. In this study, we took a mass spectrometry-based approach to identify Chlamydia effector proteins based on their interaction with Slc1, an abundant T3S chaperone. We identified a previously uncharacterized protein, Ct875/TepP, as a new T3S effector and determined that TepP is phosphorylated upon translocation into host cells, leading to the recruitment of the host scaffolding protein Crk and presumably manipulating Crk-dependent signaling functions. Finally, we provide genetic confirmation of the role of TepP in recruiting Crk and in modulating the expression of genes involved in innate immune responses to Chlamydia. This study is the first example of genetic validation of the function of a T3S effector in Chlamydia and a new example of a bacterial effector that directly co-opts the oncoprotein Crk to modulate host cell signaling events.

Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with “non-self” PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on “self” organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of “self” IRGM proteins from these structures., Author Summary Cell-autonomous host defense pathways directed against vacuolar pathogens constitute an essential arm of the mammalian innate immune defense system. Underlying most of these defense strategies is the ability of the host cell to recognize foreign or pathogen-modified structures and to deliver antimicrobial molecules specifically to these sites. Specific targeting of molecules to pathogen-containing vacuoles (PVs) requires host cells to recognize PVs as “non-self” structures that are distinct from intact “self” structures like organelles and other endomembrane components. In this work, we develop a new framework for understanding a critical principle that guides the mammalian immune system in the recognition of PVs as “non-self” structures. Our data indicates that so-called IRGM proteins function as markers of “self” compartments. We find that IRGM proteins act as “guards” that prevent a set of antimicrobial GTPases from stable association with “self” membranes. Because IRGM proteins are largely absent from “non-self” PVs, we propose that intracellular immune recognition of PVs can occur via the missing of “self” IRGM proteins.

Cholera toxin (CT) gene-negative Vibrio cholerae non-O1, non-O139 strains may cause severe diarrhea though their pathogenic mechanism remains unclear. V. cholerae cytolysin (VCC) is a pore-forming exotoxin encoded in the hlyA gene of V. cholerae whose contribution to the pathogenesis is not fully understood. In this work, the virulence properties of a CT gene-negative V. cholerae non-O1, non-O139 strain causing a cholera-like syndrome were analyzed. Inoculation of rabbit ileal loops with the wild type strain induced extensive fluid accumulation, accompanied by severe histopathological damage characterized by villus shortening, lymphangiectasia and focal areas of necrosis. These pathogenic effects were abrogated by mutation of the hlyA gene thus pointing out the main role of VCC in the virulence of the strain. Interestingly, this toxin was capable of triggering apoptosis in human intestinal cell lines due to its anion channel activity. Moreover, the wild type strain also induced increased apoptosis of the intestinal epithelium cells which was not observed upon inoculation of the VCC null mutant strain, indicating that VCC may trigger apoptotic cell death during infection in vivo. Altogether, these results support a main role of VCC in the pathogenesis of the CT gene-negative V. cholerae non-O1, non-O139 strain and identify apoptosis as a previously unrecognized cell death pathway triggered by VCC.

In the study presented here, the genetic characteristics of methicillin-susceptible Staphylococcus aureus (MSSA) strains isolated from patients attending hospitals in the city of Cordoba, Argentina, during 1999-2002 were evaluated to determine their genetic relationship with methicillin-resistant S. aureus (MRSA) clones as part of an effort to control the potential emergence of new epidemic MRSA strains. The results showed there is a high frequency of MSSA strains carrying Panton-Valentine leukocidin genes in invasive infections in Cordoba, Argentina, particularly in those occurring in hospital settings. Panton-Valentine leukocidin genes were found in the genomic background of one clone (ST30-N pulsotype) belonging to a successful internationally distributed MSSA lineage (clonal complex 30), which is closely related to the EMRSA-16 pandemic clone. These genes were also detected in the ancestral clone (ST5-M pulsotype) of the most prevalent MRSA epidemic clone causing healthcare-associated infections in this region, known as the Cordobes/Chilean clone. The molecular characterization of circulating MSSA strains, including the detection of Panton-Valentine leukocidin genes, is thus a useful marker for investigating the evolving epidemiology of hospital- and community-acquired MRSA clones.

Since 1999, a new, epidemic, methicillin-resistant Staphylococcus aureus (MRSA) strain, named the “Cordobes clone,” has emerged in Argentina and coexists with the pandemic Brazilian clone. The purpose of this study was to determine the stability over time of the new clone and to investigate its evolutionary relationship with epidemic international MRSA lineages and with other MRSA and methicillin-susceptible S. aureus (MSSA) major clones distributed in this region. One hundred three MRSA isolates recovered in 2001 from Cordoba, Argentina, hospitals and 31 MSSA strains collected from 1999 to 2002 were analyzed by their antibiotic resistance patterns, phage typing, and pulsed-field gel electrophoresis. Additionally, representative members of most MRSA defined genotypes (A, B, C, E, K, and I) were characterized by multilocus sequence typing (MLST) and spaA and SCC mec typing. The most prevalent MSSA pulsotypes were also analyzed by MLST. Our results support the displacement of the Brazilian clone (sequence type [ST] 239, spaA type WGKAOMQ, SCC mec type IIIA) by the Cordobes clone (ST5, spaA type TIMEMDMGMGMK, SCC mec type I) in the hospital environment. MRSA and MSSA isolates shared only ST5. The data support the origin of the Cordobes clone as a member of a lineage that includes the pediatric and New York/Japan international clones and that is genetically related to the British EMRSA-3 strain. Interestingly, the pediatric clone, isolated from most community-acquired infections in Cordoba, was characterized by ST100, a single-locus variant of ST5 and a new variant of SCC mec type related to SCC mec type IVc.

The gene blaCARB-9 was located in the Vibrio cholerae super-integron, but in a different location relative to blaCARB-7. CARB-9 (pI 5.2) conferred -lactam MICs four to eight times lower than those conferred by CARB-7, differing at Ambler’s positions V97I, L124F, and T228K. Comparison of the genetic environments of all reported blaCARB genes indicated that the CARB enzymes constitute a family of cassette-encoded -lactamases. Fil: Petroni, Alejandro. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Melano, Roberto. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Saka, Héctor A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Garutti, Alicia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Mange, Laura. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Pasterán, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Rapoport, Melina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Miranda, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Faccone, Diego. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Rossi, Alicia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina. Fil: Hoffman, Paul S. Dalhousie University. Department of Microbiology and Immunology; Estados Unidos. Fil: Galas, Marcelo F. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.