First evidence for the existence of Simkania negevensis in the genitalia of human females.

INTRODUCTION: Simkania negevensis has been linked to some respiratory and non-respiratory diseases. However, there is still a serious lack of clinical investigations that attempt to determine possible body sites that could be inhabited by this microorganism and evaluate its true pathogenic capacity. The goal of this study was to examine the potential presence of Simkania and its prevalence in the genital tract of human adult females. METHODS: Lower vaginal swabs from 169 Jordanian adult females who attended Obstetrics and Gynecology clinic were collected and tested for Simkania DNA by PCR method. RESULTS: The presence of bacterial nucleic acids was confirmed in the genital system of adult females with an overall prevalence of 24.26% (41/169). Interestingly, the positivity of Simkania DNA was significantly higher in women of reproductive age than females of non-reproductive age (28.03% versus 10.81%; p≤0.05). Moreover, the presence of S. negevensis was evident in approximately 43% of females suffering from vaginal itching and/or abnormal discharge, exhibiting about two-fold increase in the positivity rate compared to detection rates assessed for women who attended the clinic for routine checkup or menstruation problems. However, the current work failed to find any link between the bacterial agent and spontaneous abortion (miscarriage). CONCLUSIONS: This study showed for the first time the presence of S. negevensis in the genitalia of human females. These novel data could provide a basis to clarify the exact role of S. negevensis in the female genitalia and its potential involvement in genital system disorders.

First evidence for the existence of Simkania negevensis in the genitalia of human females.

INTRODUCTION: Simkania negevensis has been linked to some respiratory and non-respiratory diseases. However, there is still a serious lack of clinical investigations that attempt to determine possible body sites that could be inhabited by this microorganism and evaluate its true pathogenic capacity. The goal of this study was to examine the potential presence of Simkania and its prevalence in the genital tract of human adult females. METHODS: Lower vaginal swabs from 169 Jordanian adult females who attended Obstetrics and Gynecology clinic were collected and tested for Simkania DNA by PCR method. RESULTS: The presence of bacterial nucleic acids was confirmed in the genital system of adult females with an overall prevalence of 24.26% (41/169). Interestingly, the positivity of Simkania DNA was significantly higher in women of reproductive age than females of non-reproductive age (28.03% versus 10.81%; p≤0.05). Moreover, the presence of S. negevensis was evident in approximately 43% of females suffering from vaginal itching and/or abnormal discharge, exhibiting about two-fold increase in the positivity rate compared to detection rates assessed for women who attended the clinic for routine checkup or menstruation problems. However, the current work failed to find any link between the bacterial agent and spontaneous abortion (miscarriage). CONCLUSIONS: This study showed for the first time the presence of S. negevensis in the genitalia of human females. These novel data could provide a basis to clarify the exact role of S. negevensis in the female genitalia and its potential involvement in genital system disorders.

Molecular evidence for the absence of an association between Simkania negevensis and respiratory diseases.

Simkania negevensis has been implicated in respiratory diseases. This study aimed to unveil the aetiological role of this bacterium in community-acquired pneumonia (CAP) and bronchitis in Jordanian adults. Nasopharyngeal samples were collected from 98 CAP or bronchitis patients and 96 control individuals, and tested for Simkania nucleic acids using a PCR assay. The overall prevalence of the bacterial DNA in patients was markedly high and reached 57.1 %. Intriguingly, Simkania DNA was detected in 62.5 % of the nasopharyngeal swabs collected from apparently healthy controls (P>0.05). The DNA positivity in the bronchitis and CAP subgroups was 57.7 and 56.9 %, respectively, percentages that are approximately comparable to the DNA positivity assessed for the entire patient population. Simkania is most likely not a causative agent of CAP or bronchitis, despite its remarkable high prevalence. This organism, in the nasopharynx, is potentially harmless to the host and may coexist in a commensal relationship.

Lack of strong association of Chlamydia pneumoniae and atherosclerosis in a Jordanian population.

INTRODUCTION: The correlation of Chlamydia pneumoniae to coronary artery disease (CAD) in Jordan was investigated in this study. METHODOLOGY: Totals of 361 atherosclerotic patients and 392 apparently healthy controls of both sexes were enrolled. C. pneumoniae-specific IgG antibodies were measured by the microimmunofluorescence assay (MIF). The presence of the bacterial DNA in the blood by polymerase chain reaction (PCR) was also tested. RESULTS: The overall IgG seroprevalence, estimated at a titer of 1/16, was insignificantly higher in patients (75.9%) than in controls (71.7%). About 59.3% of patients demonstrated seropositivity at titers ≤ 1/256, which are suggestive of chronic or presumed past infection, whereas 54.1% of controls were seropositive at these titers (p > 0.05). Analysis of gender-specific seroprevalences revealed no obvious relation between C. pneumoniae and atherosclerosis in males (78.9% and 77.9% in atherosclerotic and control males, respectively; p > 0.05). However, a significantly elevated seropositivity was detected in atherosclerotic females (71.7%) compared with control females (64.2%). On the other hand, the PCR-based detection of C. pneumoniae DNA failed to correlate the bacterium to atherosclerosis. CONCLUSIONS: We were unable to show a strong association between C. pneumoniae and CAD, potentially because of the presence of high seroprevalence of C. pneumoniae antibodies and the unreliability of the whole blood-based nested PCR technique used.

Role of Chlamydia pneumoniae in community-acquired pneumonia in hospitalized Jordanian adults.

INTRODUCTION: This study investigated the role of Chlamydia pneumoniae in the etiology of community-acquired pneumonia (CAP) in Jordanian adults. METHODOLOGY: Eighty hospitalized CAP patients and 110 healthy adults were enrolled. Overall prevalences of C. pneumoniae IgG antibodies in sera and the rate of acute infection were estimated, using the microimmunofluorescence method (MIF), at titers of 1:16 and 1:512, respectively. Moreover, a nested polymerase chain reaction (PCR) was used to detect C. pneumoniae DNA in nasopharyngeal and blood Buffy coat samples. RESULTS: Overall chlamydial IgG prevalence was higher in CAP cases than controls (70% versus 61.8%). Similarly, higher rate of acute infection was found in patients than in controls (16.3% versus 5.5%). By focusing on subjects testing positive at 1:16, acute infection was detectable in 23.2% of CAP cases, compared with 8.8% of seropositive controls. Chlamydial DNA was confirmed in 8.2% and 8.8% of nasopharyngeal specimens from controls and patients, respectively. Moreover, 10.9% and 7.5% of Buffy coats from controls and cases, respectively, were PCR-positive. When performances of both assays for detection of the pathogen were assessed, the sensitivities of MIF and PCR were low and comparable. However, MIF demonstrated higher specificity, positive predictive value, and negative predictive value than PCR. CONCLUSIONS: MIF-based data indicate that C. pneumoniae could be a potential causal agent of CAP in Jordan. This study may serve as a basis to elucidate the exact role C. pneumoniae and other co-infecting pathogens in the etiology of respiratory tract disease.

High seroprevalence of Simkania negevensis in Jordan

The bacterium Simkania negevensis is a germ associated with respiratory diseases. This study aims at estimating the prevalence of Simkania in the Jordanian population. Serum samples from 664 Jordanian males and females, aged 2 to 86 years were collected. IgG and IgM Simkania-specific antibodies were detected using an indirect immunofluorescence test. Seropositivity titers for IgG and IgM were defined as 1:8 and 1:10, respectively. The overall prevalence of IgG antibody in all examined Jordanian nationals was 58.4%. IgG seropositivity was low in children under the age of 10 years (34.2%), and increased rapidly with age and ranged between 49.4% and 72%. Simkania-specific IgM was detected in 24.8% of subjects. IgM prevalence in children under 10 years was lowest (10.5%) and increased in older ages and remained above 20%. Overall detection rates of both IgG and IgM were significantly higher in females than males (60.7% vs. 54.5% for IgG and 26.7% vs. 21.7% for IgM). These data indicate that Simkania infection is highly prevalent in Jordan. The high level of seropositivity is most likely maintained by re-infections or chronic infections. Our data may serve as a basis to elucidate the pathogenesis of Simkania in Jordan.

Chlamydia trachomatis remodels stable microtubules to coordinate Golgi stack recruitment to the chlamydial inclusion surface.

Chlamydia trachomatis (Ctr), an obligate intracellular bacterium, survives and replicates within a membrane-bound vacuole, termed the inclusion, which intercepts host exocytic pathways to acquire nutrients. Ctr subverts cellular trafficking pathways from the Golgi by targeting small GTPases, including Rab proteins, to sustain intracellular bacterial replication; however, the precise mechanisms involved remain incompletely understood. Here, we show that Chlamydia infection in human epithelial cells induces microtubule remodeling, in particular the formation of detyrosinated stable MTs, to recruit Golgi ministacks, but not recycling endosomes, to the inclusion. These stable microtubules show increased resistance to chemically induced depolymerization, and their selective depletion results in reduced bacterial infectivity. Rab6 knockdown reversibly prevented not only Golgi ministack formation but also detyrosinated microtubule association with the inclusion. Our data demonstrate that Chlamydia co-opts the function of stable microtubules to support its development.

High prevalence of Chlamydia pneumoniae infection in an asymptomatic Jordanian population.

BACKGROUND/PURPOSE: The bacterium Chlamydia pneumoniae is associated with respiratory diseases and nonrespiratory illnesses like atherosclerosis. This study aims to investigate the seroprevalence of immunoglobulin G (IgG) against C. pneumoniae in an asymptomatic population in Jordan and to analyze the immunity state in relation to age and sex. METHODS: Serum samples were collected from 588 apparently healthy individuals aged 2-86 years. Using the microimmunofluorescence (MIF) test, seropositivity was defined as an anti-C. pneumoniae IgG titer ≥1:16. Titers from 1:16 to 1:256 were considered indicative for a past infection, whereas 1:512 was considered diagnostic of an acute infection. RESULTS: The overall prevalence of C. pneumoniae was 54.9%. The mean seropositivity in males was slightly higher than females. The seroprevalence of infection was relatively low in children aged 2-9 years, and steadily increased to reach a plateau of 66.7% at around 30-39 years of age, which remained stable in later years. Recent infection was indicated in 14.3% of study subjects. The seropositivity was highest in males, and more frequent in adults than in children and teenagers. CONCLUSION: A high seroprevalence of C. pneumoniae in the asymptomatic population suggests that infection with this pathogen is common in Jordan. Higher seropositivity in males compared to females was observed. The primary infection is acquired during the first four decades of life, and in older ages high antibody levels are likely maintained by reinfection or persistent infection.

High seroprevalence of Simkania negevensis in Jordan.

The bacterium Simkania negevensis is a germ associated with respiratory diseases. This study aims at estimating the prevalence of Simkania in the Jordanian population. Serum samples from 664 Jordanian males and females, aged 2 to 86 years were collected. IgG and IgM Simkania-specific antibodies were detected using an indirect immunofluorescence test. Seropositivity titers for IgG and IgM were defined as 1:8 and 1:10, respectively. The overall prevalence of IgG antibody in all examined Jordanian nationals was 58.4%. IgG seropositivity was low in children under the age of 10 years (34.2%), and increased rapidly with age and ranged between 49.4% and 72%. Simkania-specific IgM was detected in 24.8% of subjects. IgM prevalence in children under 10 years was lowest (10.5%) and increased in older ages and remained above 20%. Overall detection rates of both IgG and IgM were significantly higher in females than males (60.7% vs. 54.5% for IgG and 26.7% vs. 21.7% for IgM). These data indicate that Simkania infection is highly prevalent in Jordan. The high level of seropositivity is most likely maintained by re-infections or chronic infections. Our data may serve as a basis to elucidate the pathogenesis of Simkania in Jordan.

Autophagy restricts Chlamydia trachomatis growth in human macrophages via IFNG-inducible guanylate binding proteins.

Interferon γ (IFNG) is a key host response regulator of intracellular pathogen replication, including that of Chlamydia spp The antichlamydial functions of IFNG manifest in a strictly host, cell-type and chlamydial strain dependent manner. It has been recently shown that the IFNG-inducible family of immunity-related GTPases (IRG) proteins plays a key role in the defense against nonhost adapted chlamydia strains in murine epithelial cells. In humans, IFN-inducible guanylate binding proteins (hGBPs) have been shown to potentiate the antichlamydial effect of IFNG; however, how hGBPs regulate this property of IFNG is unknown. In this study, we identified hGBP1/2 as important resistance factors against C. trachomatis infection in IFNG-stimulated human macrophages. Exogenous IFNG reduced chlamydial infectivity by 50 percent in wild-type cells, whereas shRNA hGBP1/2 knockdown macrophages fully supported chlamydial growth in the presence of exogenous IFNG. hGBP1/2 were recruited to bacterial inclusions in human macrophages upon stimulation with IFNG, which triggered rerouting of the typically nonfusogenic bacterial inclusions for lysosomal degradation. Inhibition of lysosomal activity and autophagy impaired the IFNG-mediated elimination of inclusions. Thus, hGBP1/2 are critical effectors of antichlamydial IFNG responses in human macrophages. Through their capacity to remodel classically nonfusogenic chlamydial inclusions and stimulate fusion with autophagosomes, hGBP1/2 disable a major chlamydial virulence mechanism and contribute to IFNG-mediated pathogen clearance.

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Autophagy-independent function of MAP-LC3 during intracellular propagation of Chlamydia trachomatis.

Microtubule-associated protein 1 (MAP1) light chain 3 (LC3) has proven useful as autophagosomal marker in studies on the interaction between pathogens and the host autophagic machinery. However, the function of LC3 is known to extend above and beyond its role in autophagosome formation. We previously reported that intrinsic LC3 is associated with the intracellular Chlamydia trachomatis inclusion in human epithelial cells. Here we show that LC3, most likely the cytoplasmic nonlipidated form, interacts with the C. trachomatis inclusion as a microtubule-associated protein rather than an autophagosome-associated component. In contrast, N-terminally GFP-tagged LC3 exclusively targets autophagosomes rather than chlamydial inclusions. Immunofluorescence analysis revealed an association of LC3 and MAP1 subunits A and B with the inclusion as early as 18 h post infection. Inclusion-bound LC3 was connected with the microtubular network. Depolymerization of the microtubular architecture disrupted the association of LC3/MAP1s with the inclusion. Furthermore, siRNA-mediated silencing of the MAP1 and LC3 proteins revealed their essential function in the intracellular growth of C. trachomatis. Interestingly, defective autophagy remarkably enhanced chlamydial growth, suggesting a suppressive effect of the autophagic machinery on bacterial development. However, depletion of LC3 in autophagy-deficient cells noticeably reduced chlamydial propagation. Thus, our findings demonstrate a new function for LC3, distinct from autophagy, in intracellular bacterial pathogenesis.

IFN-gamma-inducible Irga6 mediates host resistance against Chlamydia trachomatis via autophagy.

Chlamydial infection of the host cell induces Gamma interferon (IFNgamma), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNgamma-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen's elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNgamma-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNgamma, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5-/- MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNgamma-induced Atg5-/- cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6-/-) MEFs, in which chlamydial growth is enhanced, do not respond to IFNgamma even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction.

Long-term effects of natural amino acids on infection with Chlamydia trachomatis.

Supplementation of culture media with leucine, isoleucine, methionine, or phenylalanine was previously found to inhibit Chlamydia trachomatis growth in HEp-2 cells. Here, we investigated the long-term effects of these additives on C. trachomatis infection in the same cell model. Amino acid addition 30h post-infection (pi) effectively suppressed the generation of infectious progeny monitored for 10 days pi. With the exception of phenylalanine, amino acid treatment beginning at 2h pi for up to 15 days led to a complete lack of infectious progeny. Phenylalanine treatment resulted in residual minimal infectivity. In extended supplementation experiments, very small aberrant chlamydial inclusions formed, whose numbers decreased considerably over time, and the production of infectious chlamydiae could not be rescued even upon amino acid withdrawal. Interestingly, a state of chlamydial persistence was induced under these conditions, as 16S rRNA transcripts were detected throughout treatment. However, expression of several key chlamydial genes including omp1, groEL, omcB, and those functioning for chlamydial DNA replication and cytokinesis was generally very low or even undetected, particularly in monolayers treated with Leu, Ile, or Met. These data revealed a capacity of certain amino acids to eliminate infectious chlamydial progeny. Additionally, supplementation of certain amino acids resulted in the formation of a small persistent population. Extrapolating from these findings may help formulate an anti-chlamydial treatment based on nutritional elements.

Naturally occurring amino acids differentially influence the development of Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae.

The differential influence of individual amino acids on the growth of Chlamydia trachomatis versus Chlamydia (Chlamydophila) pneumoniae was investigated. Certain essential amino acids added in excess at the middle of the infection course resulted in varying degrees of abnormality in the development of the two species. If amino acids were added as early as 2 h post-infection, these effects were even more pronounced. The most effective amino acids in terms of C. trachomatis growth inhibition were leucine, isoleucine, methionine and phenylalanine. These amino acids elicited similar effects against C. pneumoniae, except methionine, which, surprisingly, showed a lower inhibitory activity. Tryptophan and valine marginally inhibited C. trachomatis growth and, paradoxically, led to a considerable enhancement of C. pneumoniae growth. On the other hand, some non-essential amino acids administered at the middle of or throughout the infection course differentially affected the development of the two species. For example, C. trachomatis growth was efficiently inhibited by glycine and serine, whereas C. pneumoniae was relatively less sensitive to these agents. Another difference was apparent for glutamate, glutamine and aspartate, which stimulated C. pneumoniae growth more than that of C. trachomatis. Overall, several distinctive patterns of susceptibility to excess amino acid levels were revealed for two representative C. trachomatis and C. pneumoniae isolates. Perturbation of amino acid levels, e.g. of leucine and isoleucine, might form a basis for the development of novel treatment or preventive regimens for chlamydial diseases.

Interaction of Chlamydia trachomatis serovar L2 with the host autophagic pathway.

Chlamydiae are obligate intracellular pathogens that replicate within a membrane-bound compartment (the inclusion) and are associated with important human diseases, such as trachoma, pneumonia, and atherosclerosis. We have examined the interaction of the host autophagic pathway with Chlamydia trachomatis serovar L2 by using the specific autophagosomal stain monodansylcadaverine, antibodies to autophagosome-associated markers, and traditionally used autophagic inhibitors, particularly 3-methyladenine and amino acids. Chlamydial inclusions did not sequester monodansylcadaverine, suggesting absence of fusion with autophagosomes. Interestingly, exposure of cultures infected for 19 h to 3-methyladenine or single amino acids until the end of infection (44 h) caused various degrees of abnormalities in the inclusion maturation and in the progeny infectivity. Incubation of host cells with chemicals throughout the entire period of infection modulated the growth of Chlamydia even more dramatically. Remarkably, autophagosomal markers MAP-LC3 and calreticulin were redistributed to the inclusion of Chlamydia, a process that appears to be sensitive to 3-methyladenine and some amino acids. The present data indicate the lack of autophagosomal fusion with the inclusion because it was devoid of monodansylcadaverine and no distinct rim of autophagosomal protein-specific staining around the inclusion could be observed. However, high sensitivity of Chlamydia to conditions that could inhibit host autophagic pathway and the close association of MAP-LC3 and calreticulin with the inclusion membrane still suggest a potential role of host autophagy in the pathogenesis of Chlamydia.