Carnosic Acid Inhibits Herpes Simplex Virus Replication by Suppressing Cellular ATP Synthesis.

Acquiring resistance against antiviral drugs is a significant problem in antimicrobial therapy. In order to identify novel antiviral compounds, the antiviral activity of eight plants indigenous to the southern region of Hungary against herpes simplex virus-2 (HSV-2) was investigated. The plant extracts and the plant compound carnosic acid were tested for their effectiveness on both the extracellular and intracellular forms of HSV-2 on Vero and HeLa cells. HSV-2 replication was measured by a direct quantitative PCR (qPCR). Among the tested plant extracts, Salvia rosmarinus (S. rosmarinus) exhibited a 90.46% reduction in HSV-2 replication at the 0.47 µg/mL concentration. Carnosic acid, a major antimicrobial compound found in rosemary, also demonstrated a significant dose-dependent inhibition of both extracellular and intracellular forms of HSV-2. The 90% inhibitory concentration (IC90) of carnosic acid was between 25 and 6.25 µg/mL. Proteomics and high-resolution respirometry showed that carnosic acid suppressed key ATP synthesis pathways such as glycolysis, citrate cycle, and oxidative phosphorylation. Inhibition of oxidative phosphorylation also suppressed HSV-2 replication up to 39.94-fold. These results indicate that the antiviral action of carnosic acid includes the inhibition of ATP generation by suppressing key energy production pathways. Carnosic acid holds promise as a potential novel antiviral agent against HSV-2.

Indoleamine 2,3-Dioxygenase Cannot Inhibit Chlamydia trachomatis Growth in HL-60 Human Neutrophil Granulocytes.

Aims: Neutrophil granulocytes are the major cells involved in Chlamydia trachomatis (C. trachomatis)-mediated inflammation and histopathology. A key protein in human intracellular antichlamydial defense is the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) which limits the growth of the tryptophan auxotroph Chlamydia. Despite its importance, the role of IDO in the intracellular defense against Chlamydia in neutrophils is not well characterized. Methods: Global gene expression screen was used to evaluate the effect of C. trachomatis serovar D infection on the transcriptome of human neutrophil granulocytes. Tryptophan metabolite concentrations in the Chlamydia-infected and/or interferon-gamma (IFNG)-treated neutrophils were measured by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results: Our results indicate that the C. trachomatis infection had a major impact on neutrophil gene expression, inducing 1,295 genes and repressing 1,510 genes. A bioinformatics analysis revealed that important factors involved in the induction of neutrophil gene expression were the interferon-related transcription factors such as IRF1-5, IRF7-9, STAT2, ICSB, and ISGF3. One of the upregulated genes was ido1, a known infection- and interferon-induced host gene. The tryptophan-degrading activity of IDO1 was not induced significantly by Chlamydia infection alone, but the addition of IFNG greatly increased its activity. Despite the significant IDO activity in IFNG-treated cells, C. trachomatis growth was not affected by IFNG. This result was in contrast to what we observed in HeLa human cervical epithelial cells, where the IFNG-mediated inhibition of C. trachomatis growth was significant and the IFNG-induced IDO activity correlated with growth inhibition. Conclusions: IDO activity was not able to inhibit chlamydial growth in human neutrophils. Whether the IDO activity was not high enough for inhibition or other chlamydial growth-promoting host mechanisms were induced in the infected and interferon-treated neutrophils needs to be further investigated.

Beneficial Immunomodulatory Effects of Fluticasone Propionate in Chlamydia pneumoniae-Infected Mice.

The associations between inhaled corticosteroid (ICS) use and pulmonary infections remains controversial. Chlamydia pneumoniae (C. pneumoniae) accounts for asthma exacerbations; however, there are no data regarding ICS effects on C. pneumoniae infections. Thus, we investigated whether fluticasone propionate (FP) or budesonide (BUD) could affect C. pneumoniae infection in vitro and in vivo, focusing on the possible mechanisms that lead to potential anti-chlamydial outcomes. We performed direct qPCR to detect C. pneumoniae growth in infected, FP-treated, and BUD-treated A549 cells. Furthermore, FP or BUD was administered by inhalation to C. pneumoniae-infected mice. The recoverable C. pneumoniae was determined by indirect immunofluorescence. Expression levels of interferon (IFN)-γ and IFN-γ inducible chemokines were assessed by qPCR. We measured the protein concentrations of IFN-γ and of other cytokines that potentially participate in the anti-chlamydial response by ELISA. We found that FP treatment suppressed Chlamydia growth in A549 cells and in mice. Higher levels of IFN-γ gene expression were observed in FP-treated mice compared to the untreated and BUD-treated mice (p < 0.0001). IFN-γ and anti-chlamydial protein MIG/CXCL9 values were significantly higher after FP inhalation. Collectively, FP, but not BUD, suppressed C. pneumoniae growth in vitro and in vivo, which was likely due to the enhanced IFN-γ related responses.

Decoding Covid-19 with the SARS-CoV-2 Genome.

PURPOSE OF REVIEW: SARS-CoV-2, the recently emerged coronavirus (CoV) that is responsible for the current global pandemic Covid-19, first appeared in late 2019 in Wuhan, China. Here, we summarise details of the SARS-CoV-2 genome to assist understanding of the emergence, evolution and diagnosis of this deadly new virus. RECENT FINDINGS: Based on high similarities in the genome sequences, the virus is thought to have arisen from SARS-like CoVs in bats but the lack of an intermediate species containing a CoV with even greater similarity has so far eluded discovery. The critical determinant of the SARS-CoV-2 genome is the spike (S) gene encoding the viral structural protein that interacts with the host cell entry receptor ACE2. The S protein is sufficiently adapted to bind human ACE2 much more readily than SARS-CoV, the most closely related human CoV. SUMMARY: Although the SARS-CoV-2 genome is undergoing subtle evolution in humans through mutation that may enhance transmission, there is limited evidence for attenuation that might weaken the virus. It is also still unclear as to the events that led to the virus' emergence from bats. Importantly, current diagnosis requires specific recognition and amplification of the SARS-CoV-2 RNA genome by qPCR, despite these ongoing viral genome changes. Alternative diagnostic procedures relying on immunoassay are becoming more prevalent.

Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis.

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium linked to ocular and urogenital infections with potentially serious sequelae, including blindness and infertility. First-line antibiotics, such as azithromycin (AZT) and doxycycline, are effective, but treatment failures have also been reported. Encapsulation of antibiotics in liposomes is considered an effective approach for improving their local effects, bioavailability, biocompatibility and antimicrobial activity. To test whether liposomes could enhance the antichlamydial action of AZT, we encapsulated AZT in different surface-charged elastic liposomes (neutral, cationic and anionic elastic liposomes) and assessed their antibacterial potential against the C. trachomatis serovar D laboratory strain as well as the clinical isolate C. trachomatis serovar F. A direct quantitative polymerase chain reaction (qPCR) method was used to measure chlamydial genome content 48 h post infection and to determine the recoverable chlamydial growth. All the liposomes efficiently delivered AZT to HeLa 229 cells infected with the laboratory Chlamydia strain, exhibiting the minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of AZT even 4-8-fold lower than those achieved with the free AZT. The tested AZT-liposomes were also effective against the clinical Chlamydia strain by decreasing MIC values by 2-fold relative to the free AZT. Interestingly, the neutral AZT-liposomes had no effect on the MBC against the clinical strain, while cationic and anionic AZT-liposomes decreased the MBC 2-fold, hence proving the potential of the surface-charged elastic liposomes to improve the effectiveness of AZT against C. trachomatis.

Identification of similar epitopes between severe acute respiratory syndrome coronavirus-2 and Bacillus Calmette-Guérin: potential for cross-reactive adaptive immunity.

OBJECTIVES: Bacillus Calmette-Guérin (BCG) vaccination has been implicated in protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and as a non-specific immunisation method against the virus. We therefore decided to investigate T-cell and B-cell epitopes within the BCG-Pasteur strain proteome for similarity to immunogenic peptides of SARS-CoV-2. METHODS: We used NetMHC 4.0 and BepiPred 2.0 epitope prediction methods for the analysis of the BCG-Pasteur proteome to identify similar peptides to established and novel SARS-CoV-2 T-cell and B-cell epitopes. RESULTS: We found 112 BCG MHC-I-restricted T-cell epitopes similar to MHC-I-restricted T-cell SARS-CoV-2 epitopes and 690 BCG B-cell epitopes similar to SARS-CoV-2 B-cell epitopes. The SARS-CoV-2 T-cell epitopes represented 16 SARS-CoV-2 proteins, and the SARS-CoV-2 B-cell epitopes represented 5 SARS-CoV-2 proteins, including the receptor binding domain of the spike glycoprotein. CONCLUSION: Altogether, our results provide a mechanistic basis for the potential cross-reactive adaptive immunity that may exist between the two microorganisms.

Chlamydia pneumoniae Influence on Cytokine Production in Steroid-Resistant and Steroid-Sensitive Asthmatics.

Medications for asthma management consisting of inhaled corticosteroids act by controlling symptoms. However, some patients do not respond to steroid treatment due to immunological factors at the cytokine level. Chlamydia pneumoniae (C. pneumoniae) infection is strongly implicated in asthma pathogenesis, causing altered immune responses. We investigated the association of C. pneumoniae serostatus with the production of certain cytokines by peripheral blood mononuclear cells (PBMCs) of steroid-resistant and -sensitive asthmatic patients. Our most important findings are the following: In the case of C. pneumoniae seropositive patients we detected pronounced spontaneous interleukin (IL)-10 secretion and, in the case of steroid-resistant patients, IL-10 secretion was at a significantly higher level as compared with in-sensitive patients (p < 0.01). Furthermore, steroid-resistant seropositive patients produced a significantly higher level of IL-10 spontaneously and under antigen stimulation as compared with steroid-resistant seronegative individuals (p < 0.05). Concerning spontaneous TNF-α secretion by C. pneumoniae seropositive asthmatics, we observed that steroid-resistant patients produced significantly more of this cytokine than steroid-sensitive patients. In the steroid-resistant patients' sera, a remarkably high MMP-9 concentration was associated with C. pneumoniae seronegativity. Our study revealed that the differences in the cytokine production in steroid-sensitive and -resistant asthmatic patients can be influenced by their C. pneumoniae serostatus.

Correlation between detergent activity and anti-herpes simplex virus-2 activity of commercially available vaginal gels.

OBJECTIVE: Herpes simplex virus-2 (HSV-2) infections are almost exclusively sexually transmitted. The presence of vaginal gels during sexual activity may have a significant positive or negative impact on viral transmission. Therefore we investigated three off-the-shelf vaginal lubricants and one pH restoring gel to evaluate their impact on HSV-2 replication. RESULTS: HeLa cells were infected with untreated virions and virions incubated with the particular gels. The accumulation of viral genomes was monitored by quantitative PCR (qPCR) method at 24 h post infection. Two of the tested gels had no significant effect on HSV-2 replication at the maximum applied concentration, while two had a strong inhibitory effect (~ 98% reduction of replication). The replication inhibitory effect was observed at various multiplicity of infection (MOI 0.4-6.4) and the two inhibitory gels were also capable of inhibiting the HSV-2 induced cytopathic effect on HeLa cells. The surface tension decreasing activity-an indication of detergent activity-was strongly correlated with the anti-HSV-2 activity of the gels (R2: 0.88). Our results indicate that off-the-shelf vaginal gels have a markedly different anti-HSV-2 activity that may influence HSV-2 transmission.

Antimicrobial Resistance Screening in Chlamydia trachomatis by Optimized McCoy Cell Culture System and Direct qPCR-Based Monitoring of Chlamydial Growth.

Obligate intracellular localization of Chlamydia trachomatis (C. trachomatis) complicates antimicrobial sensitivity testing efforts that we are so accustomed to in routine bacteriology. Cell culture systems with immunofluorescence staining, to identify cellular inclusions in the presence of various concentrations of antimicrobial drugs, are still the most pervasive techniques, but more specific and sensitive nucleic acid concentration measuring methods are increasingly being used. Here we describe how to approach antimicrobial susceptibility/resistance screening in C. trachomatis by using a McCoy cell culture system, optimized by a research group from Croatia, and direct qPCR-based monitoring of chlamydial growth, optimized by a research group from Hungary.

Indoleamine 2,3-Dioxygenase Activity in Chlamydia muridarum and Chlamydia pneumoniae Infected Mouse Lung Tissues.

Chlamydia trachomatis infections are the most prevalent sexually transmitted infections with potentially debilitating sequelae, such as infertility. Mouse models are generally used for vaccine development, to study the immune response and histopathology associated with Chlamydia infection. An important question regarding murine models is the in vivo identification of murine host genes responsible for the elimination of the murine and human Chlamydia strains. RNA sequencing of the Chlamydia muridarum infected BALB/c lung transcriptome revealed that several genes with direct antichlamydial functions were induced at the tissue level, including the already described and novel members of the murine interferon-inducible GTPase family, the CXCL chemokines CXCL9, CXCL11, immunoresponsive gene 1, nitric oxide synthase-2 (iNOS), and lipocalin-2. Indoleamine 2,3-dioxygenase 1-2 (IDO1-2) previously described potent antichlamydial host enzymes were also highly expressed in the infected murine lungs. This finding was novel, since IDO was considered as a unique human antichlamydial defense gene. Besides a lower level of epithelial cell positivity, immunohistochemistry showed that IDO1-2 proteins were expressed prominently in macrophages. Detection of the tryptophan degradation product kynurenine and the impact of IDO inhibition on Chlamydia muridarum growth proved that the IDO1-2 proteins were functionally active. IDO1-2 activity also increased in Chlamydia muridarum infected C57BL/6 lung tissues, indicating that this phenomenon is not mouse strain specific. Our study shows that the murine antichlamydial response includes a variety of highly up-regulated defense genes in vivo. Among these genes the antichlamydial effectors IDO1-2 were identified. The potential impact of murine IDO1-2 expression on Chlamydia propagation needs further investigation.

Vaginal Gel Component Hydroxyethyl Cellulose Significantly Enhances the Infectivity of Chlamydia trachomatis Serovars D and E.

The transmission of the urogenital serovars of Chlamydia trachomatis can be significantly influenced by vaginal gels. Hydroxyethyl cellulose is a commonly used gelling agent that can be found in vaginal gels. Hydroxyethyl cellulose showed a concentration-dependent growth-enhancing effect on C. trachomatis serovars D and E, with a 26.1-fold maximal increase in vitro and a 2.57-fold increase in vivo.

Growth characteristics of Chlamydia trachomatis in human intestinal epithelial Caco-2 cells.

Chlamydia trachomatis is an obligate intracellular bacterium causing infections of the eyes, urogenital and respiratory tracts. Asymptomatic, repeat and chronic infections with C. trachomatis are common in the urogenital tract potentially causing severe reproductive pathology. Animal models of infection and epidemiological studies suggested the gastrointestinal tract as a reservoir of chlamydiae and as a source of repeat urogenital infections. Thus, we investigated the growth characteristics of C. trachomatis in human intestinal epithelial Caco-2 cells and the infection-induced defensin production. Immunofluorescence staining and transmission electron microscopy showed the presence of chlamydial inclusions in the cells. Chlamydial DNA and viable C. trachomatis were recovered from Caco-2 cells in similar quantity compared to that detected in the usual in vitro host cell of this bacterium. The kinetics of expression of selected C. trachomatis genes in Caco-2 cells indicated prolonged replication with persisting high expression level of late genes and of heat shock protein gene groEL. Replication of C. trachomatis induced moderate level of ß-defensin-2 production by Caco-2 cells, which might contribute to avoidance of immune recognition in the intestine. According to our results, Caco-2 cells support C. trachomatis replication, suggesting that the gastrointestinal tract is a site of residence for these bacteria.

N-acetyl-cysteine increases the replication of Chlamydia pneumoniae and prolongs the clearance of the pathogen from mice.

Purpose. Within the community, 10 % of acquired pneumonia is caused by Chlamydia pneumoniae. N-acetyl-cysteine (NAC) is one of the most commonly used mucolytics in respiratory diseases, but its effect on C. pneumoniae infection has not yet been investigated. In this study, our aim was to investigate whether NAC influences the replication of C. pneumoniae. After determining that NAC does have an effect on C. pneumoniae replication, the effect of an alternative drug called Ambroxol (Ax) was investigated.Methodology. The in vitro effect of NAC and Ax was studied on C. pneumoniae-infected A549 and McCoy cells. Furthermore, the influence of NAC and Ax was examined in mice infected intranasally with C. pneumoniae.Results. NAC treatment resulted in approximately sixfold more efficient C. pneumoniae growth in tissue culture compared to the untreated control cells, and this effect was shown to be based on the increased binding of the bacterium to the host cells. The C. pneumoniae-infected mice to which NAC was given had prolonged and more severe infections than the control mice. Ax decreased C. pneumoniae replication in vitro, which was partially associated with the increased expression of indolamine 2,3-dioxygenase. In animals, using the adapted usual human dose, Ax did not alter the number of recoverable C. pneumoniae.Conclusion. Based on our results, it might be recommended that a mucolytic agent other than NAC, such as Ax, be used in respiratory diseases suspected to be caused by C. pneumoniae.

Assembly and use of high-density recombinant peptide chips for large-scale ligand screening is a practical alternative to synthetic peptide libraries.

BACKGROUND: Recombinant peptide chips could constitute a versatile complementation to state-of-the-art in situ (chemical on-chip) synthesis, particle-based printing, or pre-manufactured peptide spotting. Bottlenecks still impeding a routine implementation - from restricted peptide lengths, low diversity and low array densities to high costs - could so be overcome. METHODS: To assess overall performance, we assembled recombinant chips composed of 38,400 individual peptide spots on the area of a standard 96-well microtiter plate from comprehensive, highly diverse (>107 single clones) short random peptide libraries. RESULTS: Screening of altogether 476,160 clones against Streptavidin uncovered 2 discrete new binders: a characteristic HPQ-motif containing VSHPQAPF and a cyclic CSGSYGSC peptide. Interactions were technically confirmed by fluorescence polarization as well as biolayer-interferometry, and their potential suitability as novel detection tags evaluated by detection of a peptide-fused exemplary test protein. CONCLUSION: From our data we conclude that the presented technical pipeline can reliably identify novel hits, useful as first-generation binders or templates for subsequent ligand design plus engineering.

A direct quantitative PCR-based measurement of herpes simplex virus susceptibility to antiviral drugs and neutralizing antibodies.

Herpes simplex viruses (HSV) are common human pathogens that can cause painful but benign manifestations and recurrent complaints, but can also cause significant morbidity and mortality on infection of the eye or brain and with disseminated infection of an immunosuppressed patient or a neonate. HSV growth inhibition measurement by plaque or yield reduction is a key task in the development of novel antiviral compounds but the manual methods are very labour intensive. The sensitive and specific PCR technology could be an effective method for quantitation of HSV DNA related to virus replication; however the currently described PCR approaches have a major limitation, namely the requirement of purification of DNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. The monitoring of HSV specific antibody titre is essential in vaccination trials and in the improvement of HSV-based oncolytic virotherapy. Usually, conventional cytopathic effect-based and plaque reduction neutralization tests are applied to measure the neutralization titre, but these methods are also time-consuming. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of HSV-2 DNA directly from the infected cells (direct qPCR) and the method was further adapted to measure the titre of HSV specific neutralizing antibody in human sera. The conditions of direct qPCR assay were optimized to measure the antiviral activity of known and novel antiviral substances. Using HSV-2 seronegative and seropositive patients' sera, the validity of the direct qPCR neutralization test was compared to traditional cytopathic effect-based assay. The direct qPCR method was able to detect the HSV-2 DNA quantitatively between multiplicity of infection 1/64 and 1/4194304, indicating that the dynamic range of the detection was approximately 65,500 fold with high correlation between the biological and technical replicates. As a proof of the adaptability of the method, we applied the direct qPCR for antiviral inhibitory concentration 50 (IC50) measurements of known and novel antiviral compounds. The measured IC50 of acyclovir was ∼0.28µg/ml, similar to the previously published IC50 value. The IC50 of novel antiviral candidates was between 1.6-3.1µg/ml. The direct qPCR-based neutralization titres of HSV positive sera were 1:32-1:64, identical to the neutralization titres determined using a traditional neutralization assay. The negative sera did not inhibit the HSV-2 replication in either of the tests. Our direct qPCR method for the HSV-2 growth determination of antiviral IC50 and neutralization titre is less time-consuming, less subjective and a more accurate alternative to the traditional plaque titration and growth reduction assays.

High dynamic range detection of Chlamydia trachomatis growth by direct quantitative PCR of the infected cells.

Chlamydiae are obligate intracellular bacteria developing in an intracytoplasmic niche, the inclusion. Chlamydia growth measurement by inclusion counting is a key task in the development of novel antichlamydial antibiotics and in vaccine studies. Most of the current counting methods rely on the immunofluorescent staining of the inclusions and either manual or automatic microscopy detection and enumeration. The manual method is highly labor intensive, while the automatic methods are either medium-throughput or require automatic microscopy. The sensitive and specific PCR technology could be an effective method for growth related chlamydial DNA detection; however the currently described PCR approaches have a major limitation, the requirement of purification of DNA or RNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of Chlamydia trachomatis DNA directly from the infected HeLa cells. With our method we were able to detect the bacterial growth in a 4 log scale (multiplicity of infection (MOI): 64 to 0.0039), with high correlation between the biological and technical replicates. As a further proof of the method, we applied the direct qPCR for antibiotic minimum inhibitory concentration (MIC) measurements. The measured MICs of moxifloxacin, tetracycline, clarithromycin and compound PCC00213 were 0.031 µg/ml, 0.031 µg/ml, 0.0039 µg/ml and 6.2 µg/ml respectively, identical or close to the already published MIC values. Our direct qPCR method for chlamydial growth and antibiotic MIC determination is less time-consuming, more objective and more sensitive than the currently applied manual or automatic fluorescent microscopy- based methods.

Protection promoted by pGP3 or pGP4 against Chlamydia muridarum is mediated by CD4(+) cells in C57BL/6N mice.

Urogenital tract infection with Chlamydia trachomatis is a leading cause of sexually transmitted infections. There is currently no commercially available vaccine against C. trachomatis. The highly conserved plasmid of chlamydiae has been considered to be a virulence factor and the plasmid proteins have important roles in the Chlamydia-specific immune response. This study was designed to evaluate the efficacy of vaccination with plasmid proteins in the prevention of C. muridarum lung infection in a mouse model. C57BL/6N mice were immunised 3 times subcutaneously with recombinant pGP3 or pGP4 and infected with C. muridarum. Immunisation of the mice with recombinant pGP3 or pGP4 protein caused a significantly lower chlamydial burden in the lungs of the infected mice; the lower IFN-γ level indicated a reduced extent of inflammation. In vitro or in vivo neutralisation of C. muridarum with sera obtained from immunised mice did not reduce the number of viable C. muridarum in the lungs of mice. However, adoptive transfer of the CD4(+) spleen cells isolated from the immunised mice resulted in a significantly reduced bacterial burden. Our results indicate that it is not the pGP3- and pGP4-specific antibodies, but the CD4(+) cells that are responsible for the protective effect of the immune response to plasmid proteins.

Anti-chlamydial effect of plant peptides.

Even in asymptomatic cases of Chlamydia trachomatis infection, the aim of the antibiotic strategy is eradication of the pathogen so as to avoid the severe late sequelae, such as pelvic inflammatory disease, ectopic pregnancy, and tubal infertility. Although first-line antimicrobial agents have been demonstrated to be predominantly successful in the treatment of C. trachomatis infection, treatment failures have been observed in some cases. Rich source of antimicrobial peptides was recently discovered in Medicago species, which act in plants as differentiation factors of the endosymbiotic bacterium partner. Several of these symbiotic plant peptides have proved to be potent killers of various bacteria in vitro. We show here that 7 of 11 peptides tested exhibited antimicrobial activity against C. trachomatis D, and that the killing activity of these peptides is most likely due to their interaction with specific bacterial targets.

Expression of Chlamydia muridarum plasmid genes and immunogenicity of pGP3 and pGP4 in different mouse strains.

Chlamydia muridarum carries a cryptic plasmid (pMoPn) of 7.5kb, which encodes seven genes. Our aims were to describe the transcriptional pattern of the pMoPn genes in C. muridarum-infected mice and to evaluate the host immune responses against pGP3 and pGP4 proteins. BALB/c and C57BL/6N female mice were inoculated intranasally with C. muridarum and sacrificed at different time points, and the total RNA was extracted from the lung suspensions to determine the levels of expression of the different plasmid genes by RT qPCR. The supernatants of the lungs were subjected to the quantitation of recoverable C. muridarum. TCA04 and TCA05, which encode pGP3 and pGP4, respectively, were amplified by PCR and cloned into the pET vector. The proteins were overexpressed in E. coli HB101 and purified. Selected groups of BALB/c and C57BL/6N mice were infected with C. muridarum 1-3 times. The humoral immune responses in the sera of the mice to the proteins encoded by TCA04 and TCA05 were tested by Western blotting, and the cellular immune responses were assessed in lymphocyte proliferation assays. The proteins recognized by the mouse sera were further analysed by a LC/MSMS technique. The kinetics of C. muridarum growth were similar in the mouse strains used, but the pathogen burden was higher in the BALB/c mice in the late phase of infection. All the plasmid genes in the BALB/c mice showed an increased level of expression on day 7, whereas the expression of the same genes did not change on day 7 in the C57BL/6N mice. The levels of expression of the plasmid genes were higher in the C57BL/6N mice at later time points. In Western blot assays, the sera of the singly infected C57BL/6N mice reacted with the monomeric form of pGP3, whereas the sera of the singly infected BALB/c mice reacted with the trimeric form of pGP3. The sera of the multiply infected C57BL/6N mice also recognized pGP4. Similarly to the humoral immune response, cellular immune responses to pGP3 and pGP4 were detected in the C. muridarum-infected C57BL/6N mice, but the spleen cells of BALB/c mice responded with proliferation only to the pGP3 protein. These results suggest that the proteins encoded by pMoPn genes may modulate the host immune response during C. muridarum infection, and that the evolved immune response against plasmid proteins, similarly to that against other chlamydial proteins, depends on the genetic background of the host.

Application of DNA chip scanning technology for automatic detection of Chlamydia trachomatis and Chlamydia pneumoniae inclusions.

Chlamydiae are obligate intracellular bacteria that propagate in the inclusion, a specific niche inside the host cell. The standard method for counting chlamydiae is immunofluorescent staining and manual counting of chlamydial inclusions. High- or medium-throughput estimation of the reduction in chlamydial inclusions should be the basis of testing antichlamydial compounds and other drugs that positively or negatively influence chlamydial growth, yet low-throughput manual counting is the common approach. To overcome the time-consuming and subjective manual counting, we developed an automatic inclusion-counting system based on a commercially available DNA chip scanner. Fluorescently labeled inclusions are detected by the scanner, and the image is processed by ChlamyCount, a custom plug-in of the ImageJ software environment. ChlamyCount was able to measure the inclusion counts over a 1-log-unit dynamic range with a high correlation to the theoretical counts. ChlamyCount was capable of accurately determining the MICs of the novel antimicrobial compound PCC00213 and the already known antichlamydial antibiotics moxifloxacin and tetracycline. ChlamyCount was also able to measure the chlamydial growth-altering effect of drugs that influence host-bacterium interaction, such as gamma interferon, DEAE-dextran, and cycloheximide. ChlamyCount is an easily adaptable system for testing antichlamydial antimicrobials and other compounds that influence Chlamydia-host interactions.