Ureaplasma urealyticum and Mycoplasma hominis urogenital infections associate with semen inflammation and decreased sperm quality.

Ureaplasma urealyticum and Mycoplasma hominis are among the most prevalent sexually transmitted infections proposed to induce urogenital inflammation and impair sperm quality. However, the topic remains controversial since contradictory findings have been reported. Herein, we performed a comprehensive analysis of U. urealyticum and M. hominis urogenital infections and their association with urogenital inflammation (i.e., leukocyte subsets and inflammatory cytokines in semen,) and sperm quality parameters in a cohort of men with couple's primary infertility undergoing initial infertility evaluation or with lower urinary tract symptoms and no infertility-related complaints. Overall, U. urealyticum and M. hominis infection was detected in 17.0% and 23.6% of patients, respectively, whereas the coinfection was detected in 3.8% of patients only. Remarkably, similar infection frequencies were found in the different patient subpopulations analyzed. Moreover, infections were associated with elevated semen levels of TNF, IL-1ß, and IL-6 and/or increased counts of total leukocytes and their subsets, including CD4 and CD8 T lymphocytes and neutrophils. In addition, M. hominis infection and the coinfection with U. urealyticum were associated with impairments in sperm quality variables. Our results indicate that U. urealyticum and M. hominis male urogenital infections induce urogenital inflammation and decrease sperm quality, thus impairing male fertility potential. Screening for U. urealyticum and M. hominis infections and performing a comprehensive analysis of different leukocyte subsets and inflammatory cytokines in semen may be clinically helpful in the diagnosis and follow-up of male urogenital infection.

Impedimetric and amperometric genosensors for the highly sensitive quantification of SARS-CoV-2 nucleic acid using an avidin-functionalized multi-walled carbon nanotubes biocapture platform.

We report two novel genosensors for the quantification of SARS-CoV-2 nucleic acid using glassy carbon electrodes modified with a biocapture nanoplatform made of multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with avidin (Av) as a support of the biotinylated-DNA probes. One of the genosensors was based on impedimetric transduction offering a non-labelled and non-amplified detection of SARS-CoV-2 nucleic acid through the increment of [Fe(CN)6]3-/4- charge transfer resistance. This biosensor presented an excellent analytical performance, with a linear range of 1.0 × 10-18 M - 1.0 × 10-11 M, a sensitivity of (5.8 ± 0.6) x 102 Ω M-1 (r2 = 0.994), detection and quantification limits of 0.33 aM and 1.0 aM, respectively; and reproducibilities of 5.4% for 1.0 × 10-15 M target using the same MWCNTs-Av-bDNAp nanoplatform, and 6.9% for 1.0 × 10-15 M target using 3 different nanoplatforms. The other genosensor was based on a sandwich hybridization scheme and amperometric transduction using the streptavidin(Strep)-biotinylated horseradish peroxidase (bHRP)/hydrogen peroxide/hydroquinone (HQ) system. This genosensor allowed an extremely sensitive quantification of the SARS-CoV-2 nucleic acid, with a linear range of 1.0 × 10-20 M - 1.0 × 10-17 M, detection limit at zM level, and a reproducibility of 11% for genosensors prepared with the same MWCNTs-Av-bDNAp1 nanoplatform. As a proof-of-concept, and considering the extremely high sensitivity, the genosensor was challenged with highly diluted samples obtained from SARS-CoV-2 RNA PCR amplification.

Ptr/CTL0175 Is Required for the Efficient Recovery of Chlamydia trachomatis From Stress Induced by Gamma-Interferon.

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 IFNγ. 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.

c-Jun Proto-Oncoprotein Plays a Protective Role in Lung Epithelial Cells Exposed to Staphylococcal α-Toxin.

c-Jun is a member of the early mammalian transcriptional regulators belonging to the AP-1 family, which participates in a wide range of cellular processes such as proliferation, apoptosis, tumorigenesis, and differentiation. Despite its established role in cell survival upon stress, its participation in the stress response induced by bacterial infections has been poorly investigated. To study the potential role of c-Jun in this context we choose the widely studied α-toxin produced by Staphylococcus aureus, a pore-forming toxin that is a critical virulence factor in the pathogenesis of these bacteria. We analyzed the effect of α-toxin treatment in the activation, expression, and protein levels of c-Jun in A549 lung epithelial cells. Furthermore, we explored the role of c-Jun in the cellular fate after exposure to α-toxin. Our results show that staphylococcal α-toxin per se is able to activate c-Jun by inducing phosphorylation of its Serine 73 residue. Silencing of the JNK (c-Jun N-terminal Kinase) signaling pathway abrogated most of this activation. On the contrary, silencing of the ERK (Extracellular Signal-Regulated Kinase) pathway exacerbated this response. Intriguingly, while the exposure to α-toxin induced a marked increase in the levels of c-Jun transcripts, c-Jun protein levels noticeably decreased in the same time-frame as a consequence of active proteolytic degradation through the proteasome-dependent pathway. In addition, we established that c-Jun promoted cell survival when cells were challenged with α-toxin. Similarly, c-Jun phosphorylation was also induced in cells upon intoxication with the cytolysin produced by Vibrio cholerae in a JNK-dependent manner, suggesting that c-Jun-JNK axis would be a conserved responsive cellular pathway to pore-forming toxins. This study contributes to understanding the role of the multifaceted c-Jun proto-oncoprotein in cell response to bacterial pore-forming toxins, positioning it as a relevant component of the complex early machinery mounted to deal with staphylococcal infections.

Erratum: Chlamydia trachomatis neither exerts deleterious effects on spermatozoa nor impairs male fertility.

A correction to this article has been published and is linked from the HTML version of this paper. The error has not been fixed in the paper.

Chlamydia trachomatis neither exerts deleterious effects on spermatozoa nor impairs male fertility.

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.

New patterns of methicillin-resistant Staphylococcus aureus (MRSA) clones, community-associated MRSA genotypes behave like healthcare-associated MRSA genotypes within hospitals, Argentina.

Methicillin-resistant Staphylococcus aureus (MRSA) burden is increasing worldwide in hospitals [healthcare-associated (HA)-MRSA] and in communities [community-associated (CA)-MRSA]. However, the impact of CA-MRSA within hospitals remains limited, particularly in Latin America. A countrywide representative survey of S. aureus infections was performed in Argentina by analyzing 591 clinical isolates from 66 hospitals in a prospective cross-sectional, multicenter study (Nov-2009). This work involved healthcare-onset infections-(HAHO, >48 hospitalization hours) and community-onset (CO) infections [including both, infections (HACO) in patients with healthcare-associated risk-factors (HRFs) and infections (CACO) in those without HRFs]. MRSA strains were genetically typed as CA-MRSA and HA-MRSA genotypes (CA-MRSAG and HA-MRSAG) by SCCmec- and spa-typing, PFGE, MLST and virulence genes profile by PCR. Considering all isolates, 63% were from CO-infections and 55% were MRSA [39% CA-MRSAG and 16% HA-MRSAG]. A significantly higher MRSA proportion among CO- than HAHO-S. aureus infections was detected (58% vs 49%); mainly in children (62% vs 43%). The CA-MRSAG/HA-MRSAG have accounted for 16%/33% of HAHO-, 39%/13% of HACO- and 60.5%/0% of CACO-infections. Regarding the epidemiological associations identified in multivariate models for patients with healthcare-onset CA-MRSAG infections, CA-MRSAG behave like HA-MRSAG within hospitals but children were the highest risk group for healthcare-onset CA-MRSAG infections. Most CA-MRSAG belonged to two major clones: PFGE-type N-ST30-SCCmecIVc-t019-PVL(+) and PFGE-type I-ST5-IV-SCCmecIVa-t311-PVL(+) (45% each). The ST5-IV-PVL(+)/ST30-IV-PVL(+) clones have caused 31%/33% of all infections, 20%/4% of HAHO-, 43%/23% of HACO- and 35%/60% of CACO- infections, with significant differences by age groups (children/adults) and geographical regions. Importantly, an isolate belonging to USA300-0114-(ST8-SCCmecIVa-spat008-PVL(+)-ACME(+)) was detected for the first time in Argentina. Most of HA-MRSAG (66%) were related to the Cordobes/Chilean clone-(PFGE-type A-ST5-SCCmecI-t149) causing 18% of all infections (47% of HAHO- and 13% of HACO-infections). Results strongly suggest that the CA-MRSA clone ST5-IV-PVL(+) has begun to spread within hospitals, replacing the traditional Cordobes/Chilean-HA-MRSA clone ST5-I-PVL(-), mainly in children. Importantly, a growing MRSA reservoir in the community was associated with spreading of two CA-MRSA clones: ST5-IV-PVL(+), mainly in children with HRFs, and ST30-IV-PVL(+) in adults without HRFs. This is the first nationwide study in Argentina providing information about the molecular and clinical epidemiology of CA-MRSA, particularly within hospitals, which is essential for designing effective control measures in this country and worldwide.

Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

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.

Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches.

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.

The Chlamydia trachomatis type III secretion chaperone Slc1 engages multiple early effectors, including TepP, a tyrosine-phosphorylated protein required for the recruitment of CrkI-II to nascent inclusions and innate immune signaling.

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.

IRG and GBP host resistance factors target aberrant, "non-self" vacuoles characterized by the missing of "self" IRGM proteins.

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.

Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms.

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.

Emergence and dissemination of a community-associated methicillin-resistant Panton-Valentine leucocidin-positive Staphylococcus aureus clone sharing the sequence type 5 lineage with the most prevalent nosocomial clone in the same region of Argentina.

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.

The autophagic pathway: a cell survival strategy against the bacterial pore-forming toxin Vibrio cholerae cytolysin.

Vibrio cholerae is the causative agent of cholera in humans. In addition to the criticalvirulence factors cholera toxin and toxin coregulated pilus, V. cholerae secretes V.cholerae cytolysin (VCC), a pore-forming exotoxin able to induce cell lysis and extensivevacuolation. We have shown that this vacuolation is related to the activation of autophagyin response to VCC action. Furthermore, we found that the autophagic pathway wasrequired to protect cells upon VCC intoxication. Based on additional data presented here,we propose a model aimed to explain the mechanism of cell protection. We postulatethat VCC-induced autophagic vacuoles, which display features of multivesicular bodies and enclose the toxin, are implicated in cell defense through VCC degradation involvingfusion with lysosomes.

Evolution and molecular characterization of methicillin-resistant Staphylococcus aureus epidemic and sporadic clones in Cordoba, Argentina.

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 SCCmec 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, SCCmec type IIIA) by the Cordobes clone (ST5, spaA type TIMEMDMGMGMK, SCCmec 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 SCCmec type related to SCCmec type IVc.

CARB-9, a carbenicillinase encoded in the VCR region of Vibrio cholerae non-O1, non-O139 belongs to a family of cassette-encoded beta-lactamases.

The gene bla(CARB-9) was located in the Vibrio cholerae super-integron, but in a different location relative to bla(CARB-7). CARB-9 (pI 5.2) conferred beta-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 bla(CARB) genes indicated that the CARB enzymes constitute a family of cassette-encoded beta-lactamases.