Chlamydia Trachomatis Cross-Serovar Protection during Experimental Lung Reinfection in Mice

Chlamydia trachomatis causes most bacterial sexually transmitted diseases worldwide. Different major outer membrane proteins (MOMPs) define various serovars of this intracellular pathogen: In women, D to L3 can cause urethritis, cervicitis, salpingitis, and oophoritis, and, thus, infertility. Protective immunity might be serovar-specific since chlamydial infection does not appear to induce an effective acquired immunity and reinfections occur. A better understanding of induced cross-serovar protection is essential for the selection of suitable antigens in vaccine development. In our mouse lung infection screening model, we evaluated the urogenital serovars D, E, and L2 in this regard. Seven weeks after primary infection or mock-infection, respectively, mice were infected a second time with the identical or one of the other serovars. Body weight and clinical score were monitored for 7 days. Near the peak of the second lung infection, bacterial load, myeloperoxidase, IFN-γ, and TNF-α in lung homogenate, as well as chlamydia-specific IgG levels in blood were determined. Surprisingly, compared with mice that were infected then for the first time, almost independent of the serovar combination used, all acquired parameters of disease were similarly diminished. Our reinfection study suggests that efficient cross-serovar protection could be achieved by a vaccine combining chlamydial antigens that do not include nonconserved MOMP regions.

Vascular Inflammation Is Associated with Loss of Aquaporin 1 Expression on Endothelial Cells and Increased Fluid Leakage in SARS-CoV-2 Infected Golden Syrian Hamsters

Vascular changes represent a characteristic feature of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection leading to a breakdown of the vascular barrier and subsequent edema formation. The aim of this study was to provide a detailed characterization of the vascular alterations during SARS-CoV-2 infection and to evaluate the impaired vascular integrity. Groups of ten golden Syrian hamsters were infected intranasally with SARS-CoV-2 or phosphate-buffered saline (mock infection). Necropsies were performed at 1, 3, 6, and 14 days post-infection (dpi). Lung samples were investigated using hematoxylin and eosin, alcian blue, immunohistochemistry targeting aquaporin 1, CD3, CD204, CD31, laminin, myeloperoxidase, SARS-CoV-2 nucleoprotein, and transmission electron microscopy. SARS-CoV-2 infected animals showed endothelial hypertrophy, endothelialitis, and vasculitis. Inflammation mainly consisted of macrophages and lower numbers of T-lymphocytes and neutrophils/heterophils infiltrating the vascular walls as well as the perivascular region at 3 and 6 dpi. Affected vessels showed edema formation in association with loss of aquaporin 1 on endothelial cells. In addition, an ultrastructural investigation revealed disruption of the endothelium. Summarized, the presented findings indicate that loss of aquaporin 1 entails the loss of intercellular junctions resulting in paracellular leakage of edema as a key pathogenic mechanism in SARS-CoV-2 triggered pulmonary lesions.

Transcriptome analysis of wheat spikes in response to Tilletia controversa Kühn which cause wheat dwarf bunt

Wheat dwarf bunt is caused by Tilletia controversa Kühn, which is one of the most destructive diseases of wheat worldwide. To explore the interaction of T. controversa and wheat, we analysed the transcriptome profile of spikes of the susceptible wheat cultivar Dongxuan 3, which was subjected to a T. controversa infection and a mock infection. The results obtained from a differential expression analysis of T. controversa-infected plants compared with mock-infected ones showed that 10,867 out of 21,354 genes were upregulated, while 10,487 genes were downregulated, and these genes were enriched in 205 different pathways. Our findings demonstrated that the genes associated with defence against diseases, such as PR-related genes, WRKY transcription factors and mitogen-activated protein kinase genes, were more highly expressed in response to T. controversa infection. Additionally, a number of genes related to physiological attributes were expressed during infection. Three pathways were differentiated based on the characteristics of gene ontology classification. KEGG enrichment analysis showed that twenty genes were expressed differentially during the infection of wheat with T. controversa. Notable changes were observed in the transcriptomes of wheat plants after infection. The results of this study may help to elucidate the mechanism governing the interactions between this pathogen and wheat plants and may facilitate the development of new methods to increase the resistance level of wheat against T. controversa, including the overexpression of defence-related genes.

Transcriptome profiling of avian pathogenic Escherichia coli and the mouse microvascular endothelial cell line bEnd.3 during interaction

Background Avian pathogenic Escherichia coli (APEC), an important extraintestinal pathogenic E. coli, causes colibacillosis, an acute and mostly systemic disease involving multiple organ lesions such as meningitis. Meningitis-causing APEC can invade the host central nervous system by crossing the blood–brain barrier (BBB), which is a critical step in the development of meningitis. However, the bacteria-host interaction mechanism in this process remains unclear. Methods In this study, we examined E. coli and bEnd.3 cells transcriptomes during infection and mock infection to investigate the global transcriptional changes in both organisms using RNA sequencing approach. Results When APEC infected the bEnd.3 cells, several significant changes in the expression of genes related to cell junctional complexes, extracellular matrix degradation, actin cytoskeleton rearrangement, immune activation and the inflammatory response in bEnd.3 cells were observed as compared to the mock infection group. Thus, the immune activation of bEnd.3 cells indicated that APEC infection activated host defenses. Furthermore, APEC may exploit cell junction degradation to invade the BBB. In addition, amino acid metabolism and energy metabolism related genes were downregulated and the protein export pathway related genes were upregulated in APEC cultured with bEnd.3 cells, compared to that in control. Thus, APEC may encounter starvation and express virulence factors during incubation with bEnd.3 cells. Conclusion This study provides a comprehensive overview of transcriptomic changes that occur during APEC infection of bEnd.3 cells, and offers insights into the bacterial invasion strategies and the subsequent host defense mechanism.

407. The Effect of Streptococcus pneumoniae Pneumonia on Atherosclerosis

Background Clinical studies consistently find an increase in the risk of acute coronary syndrome (ACS) in the weeks following pneumonia, although the mechanisms underlying this finding are unknown. ACS most commonly occurs as a result of thrombosis at the site of ruptured atherosclerotic plaques. We hypothesized that the systemic inflammatory response to pneumococcal pneumonia leads to acute localized inflammatory changes within established atherosclerotic plaques, favoring plaque instability and rupture, thereby resulting in ACS. Methods Male ApoE-/- mice, a well-established model of atherosclerosis, were fed an atherogenic diet for 7–8 weeks before intranasal infection with Streptococcus pneumoniae or mock infection. Mice were sacrificed 2 or 8 weeks post-infection. Formalin-fixed, paraffin-embedded aortic sinus plaque sections were analyzed to assess markers of plaque vulnerability to rupture. To characterise post-pneumonic plaque macrophage phenotype, aortic sinus plaque cryosections 2 weeks post pneumonia/mock infection were immunostained for MAC-3 to identify macrophage-rich areas. These plaque regions were collected using laser capture microdissection and RNA extracted for microarray analysis. Results S. pneumoniae infection was associated with increased aortic sinus atherosclerotic plaque macrophage content (18.1 vs. 8.0%; P < 0.05) at 2 weeks post infection, but no significant difference in aortic sinus plaque burden, plaque smooth muscle or collagen content. There was no significant difference in any of these plaque vulnerability markers at 8 weeks post infection. Microarray analysis of laser capture micro-dissected plaque macrophages identified downregulation of the expression of three genes coding for specific E3 ubiquitin ligases following pneumonia. Pathway analysis identified a significant perturbation in the ubiquitin proteasome system pathway as a result. Conclusion In this murine model, pneumococcal pneumonia resulted in increased atherosclerotic plaque macrophage content, a marker of plaque instability, at 2 weeks post infection. Pneumonia may therefore lead to an increased propensity for atherosclerotic plaques to rupture soon after pneumonia, due to infiltration of macrophages into the plaque. Disclosures All authors: No reported disclosures.

Rapid and Simple Detection of Trichosporon asahii by Optimized Colony PCR

Trichosporon asahii is the major pathogen causing invasive trichosporonosis. Conventional methods of its detection are time-consuming or costly and often require complex DNA extraction and purification steps, which hinders rapid clinical diagnosis. In this study, we evaluated colony PCR, which directly uses colonies or trace clinical samples as the template for amplification, for rapid detection of T. asahii infection. Four methods, namely, direct colony, freeze-thaw, glass beads, and enzymolysis, were compared to select the best DNA extraction strategy. We subsequently designed and screened species-specific primers targeting the intergenic spacer 1 (IGS1) of the ribosomal DNA of T. asahii and used them to detect mock infection clinical samples. The species-specific colony PCR based on glass beads proved advantageous, with short procedure time (154.8 ± 0.6 min), good sensitivity (detection limit, 102 CFU/mL), and specificity for T. asahii, indicating that this method can be used for the rapid and simple identification of clinical samples of T. asahii infection.

Matrix Metalloproteinase Activity in Infections by an Encephalitic Virus, Mouse Adenovirus Type 1

ABSTRACT Mouse adenovirus type 1 (MAV-1) infection causes encephalitis in susceptible strains of mice and alters the permeability of infected brains to small molecules, which indicates disruption of the blood-brain barrier (BBB). Under pathological conditions, matrix metalloproteinases (MMPs) can disrupt the BBB through their proteolytic activity on basement membrane and tight junction proteins. We examined whether MAV-1 infection alters MMP activity in vivo and in vitro. Infected MAV-1-susceptible SJL mice had higher MMP2 and MMP9 activity in brains, measured by gelatin zymography, than mock-infected mice. Infected MAV-1-resistant BALB/c mice had MMP activity levels equivalent to those in mock infection. Primary SJL mouse brain endothelial cells (a target of MAV-1 in vivo) infected ex vivo with MAV-1 had no difference in activities of secreted MMP2 and MMP9 from mock cells. We show for the first time that astrocytes and microglia are also infected in vivo by MAV-1. Infected mixed primary cultures of astrocytes and microglia had higher levels of MMP2 and MMP9 activity than mock-infected cells. These results indicate that increased MMP activity in the brains of MAV-1-infected susceptible mice may be due to MMP activity produced by endothelial cells, astrocytes, and microglia, which in turn may contribute to BBB disruption and encephalitis in susceptible mice. IMPORTANCE RNA and DNA viruses can cause encephalitis; in some cases, this is accompanied by MMP-mediated disruption of the BBB. Activated MMPs degrade extracellular matrix and cleave tight-junction proteins and cytokines, modulating their functions. MAV-1 infection of susceptible mice is a tractable small-animal model for encephalitis, and the virus causes disruption of the BBB. We showed that MAV-1 infection increases enzymatic activity of two key MMPs known to be secreted and activated in neuroinflammation, MMP2 and MMP9, in brains of susceptible mice. MAV-1 infects endothelial cells, astrocytes, and microglia, cell types in the neurovascular unit that can secrete MMPs. Ex vivo MAV-1 infection of these cell types caused higher MMP activity than mock infection, suggesting that they may contribute to the higher MMP activity seen in vivo. To our knowledge, this provides the first evidence of an encephalitic DNA virus in its natural host causing increased MMP activity in brains.

Human Cytomegalovirus Inhibited Megakaryocytic Differentiation, Maturation and Induced Apoptosis in Vitro.

Abstract 2413 Poster Board II-390 Human cytomegalovirus (HCMV) can cause life-threatening infection in immunocomprimised individuals, such as patients undergoing intensive chemotherapy or bone marrow transplantation. Thrombocytopenia is one of the manifestations in active HCMV infection, which may be a consequence of viral suppression on megakaryopoiesis. The exact underline mechanisms remain uncertain. Our previous studies suggested that HCMV directly infects megakaryocytic progenitors and inhibits their proliferation. Colony-formation of HCMV-infected CFU-MK decreased in a dose-dependent manner (Blood, 2003, abstract). In present study, we explored the mechanisms further by using a phorbol 12-myristate 13-acetate (PMA) stimulating polyploidization to mimic the late stage of megakaryocytic differentiation and maturation in vitro. After co-culture of a megakaryocytic cell line CHRF-288-11 with HCMV AD169 experimental strain from day 0 to day 3 (multiple of infection, MOI=1), the polyploidization of megakaryocyte was determined by DNA content analysis using flow cytometry. Compared with negative control, the proportion of polyploidy (ploidy N ≥ 8) megakaryocytes decreased by 52%, 32% and 16% in HCMV-infected cell at day 3, day 6 and day 9 respectively. As a specific receptor for megakaryopoietic differentiation, the c-Mpl protein (TPO receptor) was also examined in CHRF-288-11 cell line. The proportion of c-Mpl positive cells showed a 23% decrease in HCMV-infected group in compared to the mock infection control (using ultralviolet treated HCMV) at day 5. In addition, apoptotic signals from megakaryocytic surface, cytoplasma and mitochondria were detected in HCMV-infected cells by flow cytometry with Annexin V, Caspase-3 and JC-1 assay. Compared to mock infection control at day 5, annexin-V positive cells population increased by 57%; active caspase-3 signal increased by 125% in viable cell population; and cell population with damaged mitochondial membrane showed a 5-times increase. In conclusion, our data demonstrated that: (1) HCMV inhibited megakaryocytic differentiation and maturation at late stage; (2) HCMV reduced c-Mpl positive cell population; (3) HCMV induced megakaryocytic apoptosis through intrinsic apoptotic pathway as shown by the functional alteration of mitochodial membrane, activation of caspase-3 and structural damage of outer cellular membrane. HCMV-induced thrombocytopenia is the consequence of multiple processes involving inhibition of megakaryocytic proliferation, differentiation, maturation and also increased megakaryocytic apoptosis. Disclosures: No relevant conflicts of interest to declare.