The Chlamydia-related Waddlia chondrophila encodes functional type II toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as ß-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche.IMPORTANCEThe response to adverse conditions, such as exposure to antibiotics, nutrient starvation and competition with other microorganisms, is essential for the survival of a bacterial population. TA systems are modules composed of two elements, a toxic protein and an antitoxin (protein or RNA) that counteracts the toxin. Although many aspects of TA biological functions still await to be elucidated, TAs have often been implicated in bacterial response to stress, including the response to nutrient starvation, antibiotic treatment and bacteriophage infection. TAs are ubiquitous in free-living bacteria but rare in obligate intracellular species such as chlamydiae. We identified functional TA systems in Waddlia chondrophila, a chlamydial species with a strikingly broad host range compared to other chlamydiae. Our work contributes to understand how obligate intracellular bacteria react to adverse conditions that might arise from competition with other viruses/bacteria for the same replicative niche and would threaten their ability to replicate.

Waddlia chondrophila, a Chlamydia-related bacterium, has a negative impact on human spermatozoa.

STUDY QUESTION: What is the impact of Waddlia chondrophila, an emerging Chlamydia-related bacterium associated with miscarriage, on human spermatozoa? SUMMARY ANSWER: W. chondrophila had a negative impact on human spermatozoa (decrease in viability and mitochondrial membrane potential) and was not entirely removed from infected samples by density gradient centrifugation. WHAT IS KNOWN ALREADY: Bacterial infection or colonization might have a deleterious effect on male fertility. Waddlia chondrophila was previously associated with miscarriage, but its impact on male reproductive function has never been studied. STUDY DESIGN SIZE, DURATION: An in vitro model of human spermatozoa infection was used to assess the effects of W. chondrophila infection. Controls included Chlamydia trachomatis serovar D and latex beads with similar size to bacteria. PARTICIPANTS/MATERIALS, SETTING, METHODS: Purified motile spermatozoa were infected with W. chondrophila (multiplicity of infection of 1). Immunohistochemistry combined with confocal microscopy was used to evaluate how bacteria interact with spermatozoa. The impact on physiology was assessed by monitoring cell viability, mitochondrial membrane potential and DNA fragmentation. MAIN RESULTS AND THE ROLE OF CHANCE: Using super-resolution confocal microscopy, bacteria were localized on spermatozoa surface, as well as inside the cytoplasm. Compared to controls, W. chondrophila caused a 20% increase in mortality over 72 h of incubation (P < 0.05). Moreover, higher bacterial loads significantly reduced mitochondrial membrane potential. Bacteria present on spermatozoa surface were able to further infect a cell-monolayer, indicating that sperm might vector bacteria during sexual intercourse. LIMITATIONS REASONS FOR CAUTION: The main limitation of the study is the use of an in vitro model of infection, which might be too simplistic compared to an actual infection. An animal model of infection should be developed to better evaluate the in vivo impact of W. chondrophila. WIDER IMPLICATIONS OF THE FINDINGS: Intracellular bacteria, including C. trachomatis, Mycoplasma spp. and Ureaplasma spp., are associated with male infertility. Waddlia chondrophila might represent yet another member of this group, highlighting the need for more rigorous microbiological analysis during investigations for male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work has been funded by the Department of Obstetrics and Gynecology, Lausanne University Hospital, Switzerland, and by the Swiss National Science Foundation (Grant nos. 310030-156169/1, 320030-169853/1 and 320030-169853/2 attributed to D.B.). D.B. is also supported by the 'Fondation Leenaards' through the 'Bourse pour la relève académique', by the 'Fondation Divesa' and by the 'Loterie Romande'. No conflicts of interest to declare.

Role of Waddlia chondrophila placental infection in miscarriage.

Waddlia chondrophila is an intracellular bacterium suspected to cause human and bovine abortion. We confirmed an association between antibodies against W. chondrophila and human miscarriage and identified this organism in placenta or genital tract of women who had had miscarriages. These results suggest a possible role of W. chondrophila infection in miscarriage.

Pharmacophore modelling based virtual screening and molecular dynamics identified the novel inhibitors and drug targets against Waddlia chondrophila.

Waddlia chondrophila is a possible cause of fetal death in humans. This Chlamydia-related bacterium is an emergent pathogen that causes human miscarriages and ruminant abortions, which results in financial losses. Despite the years of efforts, the underlying mechanism behind the pathogenesis of W. chondrophila is little known which hindered the development of novel treatment options. In the framework of current study, computational approaches were used to identify novel inhibitors (phytocompounds) and drug targets against W. chondrophila. At first, RNA polymerase sigma factor SigA and 3-deoxy-D-manno-octulosonic acid transferase were identified through subtractive proteomics pipeline. Afterwards, extensive docking and simulation analyses were conducted to optimize potentially novel phytocompounds by assessing their binding affinity to target proteins. A 100ns molecular dynamics simulation well complimented the compound's binding affinity and indicated strong stability of predicted compounds at the docked site. The calculation of binding free energies with MMGBSA corroborated the significant binding affinity between phytocompounds and target protein binding sites. The proposed phytocompounds may be a viable treatment option for patients infected with W. chondrophila; however, further research is required to ensure their safety.

The role of Chlamydiales in adverse pregnancy outcome: a community-based nested case-control study.

OBJECTIVES: Over the last decades, the Chlamydiales order has expanded and a new group of Chlamydia-related bacteria has emerged, covering species such as Waddlia chondrophila associated with bovine abortion. However, it is unknown whether they compromise human reproduction such as Chlamydia trachomatis. We therefore aimed to investigate the association between vaginal colonization of selected species of the Chlamydiales order with spontaneous abortion, preterm birth, and animal exposure. METHODS: Pregnant women were enrolled at the nuchal translucency scan or when admitted for suspected miscarriage at Aarhus University Hospital, Denmark. Cases were defined as spontaneous abortion <22 weeks and preterm birth <37 weeks. Controls were defined as term birth ≥37 weeks. Vaginal samples from 1203 women were assessed using C. trachomatis, W. chondrophila, and pan-Chlamydiales-specific real-time PCRs targeting the 16S rRNA gene. RESULTS: A total of 1120 women of primarily Caucasian ancestry were enrolled, including 193 spontaneous abortions, 88 preterm births, and 839 term births. After sequencing for verification, the prevalence of Chlamydiales was 3 of 193 (1.6%; 95% CI, 0.5-4.8) in women experiencing spontaneous abortion, 2 of 88 (2.3%; 95% CI, 0.6-8.9) in women with preterm birth, and 20 of 839 (2.4%; 95% CI, 1.6-3.7) in women giving birth at term. Thus, Chlamydiales infection was neither significantly associated with spontaneous abortion (OR, 0.68; 95% CI, 0.15-2.01) nor preterm birth (OR, 1.02; 95% CI, 0.15-3.60) compared with women giving birth at term. Amplicons from the pan-Chlamydiales assay revealed close sequence homology and were primarily identified as uncultured Chlamydiales bacteria. DISCUSSION: Among Danish pregnant women, the prevalence of Chlamydiales was low and not associated with adverse pregnancy outcomes.

Subtractive proteomics approach to Unravel the druggable proteins of the emerging pathogen Waddlia chondrophila and drug repositioning on its MurB protein.

Waddlia chondrophila is an emerging pathogen that has been implicated in numerous unpropitious pregnancy events in humans and ruminants. Taking into account its association with abortigenic events, possible modes of transmission, and future risk, immediate clinical measures are required to prevent widespread damage caused by this organism and hence this study. Here, a subtractive proteomics approach was employed to identify druggable proteins of W. chondrophila. Considering the essential genes, antibiotic resistance proteins, and virulence factors, 676 unique important proteins were initially identified for this bacterium. Afterward, NCBI BLASTp performed against human proteome identified 223 proteins that were further pushed into KEGG Automatic Annotation Server (KAAS) for automatic annotation. Using the information from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database 14 Waddlia specific metabolic pathways were identified with respect to humans. Analyzing the data from KAAS and KEGG databases, forty-eight metabolic pathway-dependent, and seventy metabolic pathway independent proteins were identified. Standalone BLAST search against DrugBank FDA approved drug targets revealed eight proteins that are finally considered druggable proteins. Prediction of three-dimensional structures was done for the eight proteins through homology modeling and the Ramachandran plot model showed six models as a valid prediction. Finally, virtual screening against MurB protein was performed using FDA approved drugs to employ the drug repositioning strategy. Three drugs showed promising docking results that can be used for therapeutic purposes against W. chondrophila following the clinical validation of the study.

Impact of Waddlia chondrophila infection on pregnancy in the mouse.

The intracellular bacterium Waddlia chondrophila, which belongs to the Chlamydiales order, was found to be associated with miscarriage in humans. There is little to no knowledge regarding the mode of infection, impact on the neonate and pathophysiology of this emerging bacterium. We have previously shown that W. chondrophila induces a systemic infection, organ pathology and elicits T helper type 1-associated humoral immunity in a murine model of genital infection. In the present study, we took advantage of this model of infection to evaluate the impact of this bacterium on the mouse pregnancy. We used two routes of inoculation, vaginal and intrauterine, to introduce infection before and after mating. Our results show that genital infection by W. chondrophila did not have any significant impact on gestation length and maternal weight gain, nor on the number of offspring and their weight. This observation indicates that the mouse model of infection is not suitable to study the effect of W. chondrophila on pregnancy and alternative models of infection, including in vitro ones, should be used. Moreover, an indirect immunopathological mechanism activated by this bacterium should be further explored.

Limited presence of Waddlia chondrophila in drinking water systems in the Netherlands.

Waddlia chondrophila is an emerging pathogen belonging to the order of Chlamydiales. This obligate intracellular bacterium was initially isolated from an aborted bovine fetus and is associated with adverse pregnancy outcomes in women. The ability of W. chondrophila to reside and replicate within a range of free-living amoebae implies a possible widespread environmental presence. Potential hosts of W. chondrophila are present in Dutch drinking water. This study therefore investigated the presence of W. chondrophila DNA in drinking water by analysing 59 samples from ten drinking water systems throughout the Netherlands. Samples were taken at three distances from the treatment plant, during both summer and winter. Twelve of the samples were positive, originating from two of the treatment plants, of which three samples were quantifiable.

Transcriptional Landscape of Waddlia chondrophila Aberrant Bodies Induced by Iron Starvation.

Chronic infections caused by obligate intracellular bacteria belonging to the Chlamydiales order are related to the formation of persistent developmental forms called aberrant bodies (ABs), which undergo DNA replication without cell division. These enlarged bacteria develop and persist upon exposure to different stressful conditions such as ß-lactam antibiotics, iron deprivation and interferon-γ. However, the mechanisms behind ABs biogenesis remain uncharted. Using an RNA-sequencing approach, we compared the transcriptional profile of ABs induced by iron starvation to untreated bacteria in the Chlamydia-related species Waddliachondrophila, a potential agent of abortion in ruminants and miscarriage in humans. Consistent with the growth arrest observed following iron depletion, our results indicate a significant reduction in the expression of genes related to energy production, carbohydrate and amino acid metabolism and cell wall/envelope biogenesis, compared to untreated, actively replicating bacteria. Conversely, three putative toxin-antitoxin modules were among the most up-regulated genes upon iron starvation, suggesting that their activation might be involved in growth arrest in adverse conditions, an uncommon feature in obligate intracellular bacteria. Our work represents the first complete transcriptomic profile of a Chlamydia-related species in stressful conditions and sets the grounds for further investigations on the mechanisms underlying chlamydial persistence.

Screening of Chlamydia trachomatis and Waddlia chondrophila Antibodies in Women with Tubal Factor Infertility.

Waddlia chondrophila is an emerging intracellular pathogen belonging to the order of Chlamydiales, and was previously associated with adverse pregnancy outcomes, as well as tubal factor infertility (TFI). In this study, we investigate the link between both W. chondrophila and Chlamydia trachomatis IgG seropositivity and TFI. Antibodies against both bacteria were measured in 890 serum samples of women visiting a fertility clinic. After a hysterosalpingography and/or laparoscopy, they were classified as either TFI-negative (TFI-) or TFI-positive (TFI+). The total seroprevalence was 13.4% for C. trachomatis and 38.8% for W. chondrophila. C. trachomatis antibodies were present significantly more often in the TFI+ group than in the TFI- group, while for W. chondrophila no difference could be observed. In conclusion, our study confirms the association between C. trachomatis seropositivity and TFI, but no association was found between W. chondrophila seropositivity and TFI. The high percentage of W. chondrophila seropositivity in all women attending a fertility clinic does, however, demonstrate the need for further research on this Chlamydia-like bacterium and its possible role in infertility.

Diverse Stress-Inducing Treatments cause Distinct Aberrant Body Morphologies in the Chlamydia-Related Bacterium, Waddlia chondrophila.

Chlamydiae, such as Chlamydia trachomatis and Chlamydia pneumoniae, can cause chronic infections. It is believed that persistent forms called aberrant bodies (ABs) might be involved in this process. AB formation seems to be a common trait of all members of the Chlamydiales order and is caused by distinct stress stimuli, such as ß-lactam antibiotics or nutrient starvation. While the diverse stimuli inducing ABs are well described, no comprehensive morphological characterization has been performed in Chlamydiales up to now. We thus infected mammalian cells with the Chlamydia-related bacterium Waddlia chondrophila and induced AB formation using different stimuli. Their morphology, differences in DNA content and in gene expression were assessed by immunofluorescence, quantitative PCR, and reverse transcription PCR, respectively. All stimuli induced AB formation. Interestingly, we show here for the first time that the DNA gyrase inhibitor novobiocin also caused appearance of ABs. Two distinct patterns of ABs could be defined, according to their morphology and number: (i) small and multiple ABs versus (ii) large and rare ABs. DNA replication of W. chondrophila was generally not affected by the different treatments. Finally, no correlation could be observed between specific types of ABs and expression patterns of mreB and rodZ genes.

Eukaryotic Cell Permeabilisation to Identify New Putative Chlamydial Type III Secretion System Effectors Secreted within Host Cell Cytoplasm.

Chlamydia trachomatis and Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales order. C. trachomatis is the most frequent bacterial cause of genital and ocular infections whereas W. chondrophila is an opportunistic pathogen associated with adverse pregnancy outcomes and respiratory infections. Being strictly intracellular, these bacteria are engaged in a complex interplay with their hosts to modulate their environment and create optimal conditions for completing their life cycle. For this purpose, they possess several secretion pathways and, in particular, a Type III Secretion System (T3SS) devoted to the delivery of effector proteins in the host cell cytosol. Identifying these effectors is a crucial step in understanding the molecular basis of bacterial pathogenesis. Following incubation of infected cells with perfringolysin O, a pore-forming toxin that binds cholesterol present in plasma membranes, we analysed by mass spectrometry the protein content of the host cell cytoplasm. We identified 13 putative effectors secreted by C. trachomatis and 19 secreted by W. chondrophila. Using Y. enterocolitica as a heterologous expression and secretion system, we confirmed that four of these identified proteins are secreted by the T3SS. Two W. chondrophila T3SS effectors (hypothetical proteins Wcw_0499 and Wcw_1706) were further characterised and demonstrated to be early/mid-cycle effectors. In addition, Wcw_1706 is associated with a tetratricopeptide domain-containing protein homologous to C. trachomatis class II chaperone. Furthermore, we identified a novel C. trachomatis effector, CT460 that localises in the eukaryotic nucleus when ectopically expressed in 293 T cells.

A SpoIID Homolog Cleaves Glycan Strands at the Chlamydial Division Septum.

Chlamydiales species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal peptidoglycan biosynthesis seems to be regulated by MreB actin and its membrane anchor RodZ rather than FtsZ tubulin in Chlamydiales, the mechanism of peptidoglycan remodeling is poorly understood. An amidase conserved in Chlamydiales is able to cleave peptide stems in peptidoglycan, but it is not clear how peptidoglycan glycan strands are cleaved since no classical lytic transglycosylase is encoded in chlamydial genomes. However, a protein containing a SpoIID domain, known to possess transglycosylase activity in Bacillus subtilis, is conserved in Chlamydiales We show here that the SpoIID homologue of the Chlamydia-related pathogen Waddlia chondrophila is a septal peptidoglycan-binding protein. Moreover, we demonstrate that SpoIID acts as a lytic transglycosylase on peptidoglycan and as a muramidase on denuded glycan strands in vitro As SpoIID-like proteins are widespread in nonsporulating bacteria, SpoIID might commonly be a septal peptidoglycan remodeling protein in bacteria, including obligate intracellular pathogens, and thus might represent a promising drug target.IMPORTANCEChlamydiales species are obligate intracellular bacteria and important human pathogens that have a minimal division machinery lacking the proteins that are essential for bacterial division in other species, such as FtsZ. Chlamydial division requires synthesis of peptidoglycan, which forms a ring at the division septum and is rapidly turned over. However, little is known of peptidoglycan degradation, because many peptidoglycan-degrading enzymes are not encoded by chlamydial genomes. Here we show that an homologue of SpoIID, a peptidoglycan-degrading enzyme involved in sporulation of bacteria such as Bacillus subtilis, is expressed in Chlamydiales, localizes at the division septum, and degrades peptidoglycan in vitro, indicating that SpoIID is not only involved in sporulation but also likely implicated in division of some bacteria.

Interactions Screenings Unearth Potential New Divisome Components in the Chlamydia-Related Bacterium, Waddlia chondrophila.

Chlamydiales order members are obligate intracellular bacteria, dividing by binary fission. However, Chlamydiales lack the otherwise conserved homologue of the bacterial division organizer FtsZ and certain division protein homologues. FtsZ might be functionally replaced in Chlamydiales by the actin homologue MreB. RodZ, the membrane anchor of MreB, localizes early at the division septum. In order to better characterize the organization of the chlamydial divisome, we performed co-immunoprecipitations and yeast-two hybrid assays to study the interactome of RodZ, using Waddlia chondrophila, a potentially pathogenic Chlamydia-related bacterium, as a model organism. Three potential interactors were further investigated: SecA, FtsH, and SufD. The gene and protein expression profiles of these three genes were measured and are comparable with recently described division proteins. Moreover, SecA, FtsH, and SufD all showed a peripheral localization, consistent with putative inner membrane localization and interaction with RodZ. Notably, heterologous overexpression of the abovementioned proteins could not complement E. coli mutants, indicating that these proteins might play different functions in these two bacteria or that important regulators are not conserved. Altogether, this study brings new insights to the composition of the chlamydial divisome and points to links between protein secretion, degradation, iron homeostasis, and chlamydial division.

The role of peptidoglycan in chlamydial cell division: towards resolving the chlamydial anomaly.

Chlamydiales are obligate intracellular bacteria including some important pathogens causing trachoma, genital tract infections and pneumonia, among others. They share an atypical division mechanism, which is independent of an FtsZ homologue. However, they divide by binary fission, in a process inhibited by penicillin derivatives, causing the formation of an aberrant form of the bacteria, which is able to survive in the presence of the antibiotic. The paradox of penicillin sensitivity of chlamydial cells in the absence of detectable peptidoglycan (PG) was dubbed the chlamydial anomaly, since no PG modified by enzymes (Pbps) that are the usual target of penicillin could be detected in Chlamydiales. We review here the recent advances in this field with the first direct and indirect evidences of PG-like material in both Chlamydiaceae and Chlamydia-related bacteria. Moreover, PG biosynthesis is required for proper localization of the newly described septal proteins RodZ and NlpD. Taken together, these new results set the stage for a better understanding of the role of PG and septal proteins in the division mechanism of Chlamydiales and illuminate the long-standing chlamydial anomaly. Moreover, understanding the chlamydial division mechanism is critical for the development of new antibiotics for the treatment of chlamydial chronic infections.

Waddlia chondrophila and Chlamydia trachomatis antibodies in screening infertile women for tubal pathology.

Since Waddlia chondrophila is closely related to Chlamydia trachomatis, we hypothesise that W. chondrophila may also be associated with tubal factor infertility (TFI) in women, a major complication of chronic C. trachomatis infection. Five hundred twenty serum samples were tested for anti-Waddlia antibodies by ELISA. Among the 520 investigated women, a total number of 142 (27.3%) has had laparoscopic diagnosis performed, and were either classified TFI positive or negative. Presence of high titres of W. chondrophila antibodies was linked to TFI (p < 0.0001; OR: 7.5; 95% CI: 3.3-17). Moreover, antibody positivity to both W. chondrophila and C. trachomatis-MOMP was strongly associated with TFI (p < 0.0001; OR: 21; 95% CI: 3.8-12E1). This association was much stronger than the statistical association of C. trachomatis-MOMP antibodies only (p < 0.0001; OR: 7.1; 95% CI: 3.7-14), suggesting that co-infection with W. chondrophila and C. trachomatis may lead to more severe reproductive sequelae and immune responses than single infection with either Chlamydiales members.

Disassembly of a Medial Transenvelope Structure by Antibiotics during Intracellular Division.

Chlamydiales possess a minimal but functional peptidoglycan precursor biosynthetic and remodeling pathway involved in the assembly of the division septum by an atypical cytokinetic machine and cryptic or modified peptidoglycan-like structure (PGLS). How this reduced cytokinetic machine collectively coordinates the invagination of the envelope has not yet been explored in Chlamydiales. In other Gram-negative bacteria, peptidoglycan provides anchor points that connect the outer membrane to the peptidoglycan during constriction using the Pal-Tol complex. Purifying PGLS and associated proteins from the chlamydial pathogen Waddlia chondrophila, we unearthed the Pal protein as a peptidoglycan-binding protein that localizes to the chlamydial division septum along with other components of the Pal-Tol complex. Together, our PGLS characterization and peptidoglycan-binding assays support the notion that diaminopimelic acid is an important determinant recruiting Pal to the division plane to coordinate the invagination of all envelope layers with the conserved Pal-Tol complex, even during osmotically protected intracellular growth.

Undressing of Waddlia chondrophila to enrich its outer membrane proteins to develop a new species-specific ELISA.

Waddlia chondrophila, an obligate intracellular bacterium of the Chlamydiales order, is considered as an agent of bovine abortion and a likely cause of miscarriage in humans. Its role in respiratory diseases was questioned after the detection of its DNA in clinical samples taken from patients suffering from pneumonia or bronchiolitis. To better define the role of Waddlia in both miscarriage and pneumonia, a tool allowing large-scale serological investigations of Waddlia seropositivity is needed. Therefore, enriched outer membrane proteins of W. chondrophila were used as antigens to develop a specific ELISA. After thorough analytical optimization, the ELISA was validated by comparison with micro-immunofluorescence and it showed a sensitivity above 85% with 100% specificity. The ELISA was subsequently applied to human sera to specify the role of W. chondrophila in pneumonia. Overall, 3.6% of children showed antibody reactivity against W. chondrophila but no significant difference was observed between children with and without pneumonia. Proteomic analyses were then performed using mass spectrometry, highlighting members of the outer membrane protein family as the dominant proteins. The major Waddlia putative immunogenic proteins were identified by immunoblot using positive and negative human sera. The new ELISA represents an efficient tool with high throughput applications. Although no association with pneumonia and Waddlia seropositivity was observed, this ELISA could be used to specify the role of W. chondrophila in miscarriage and in other diseases.

Waddlia chondrophila: from biology to pathogenicity.

Waddlia chondrophila is an emerging pathogen causing miscarriages in humans and abortions in ruminants. The full genome of this Chlamydia-related bacterium has been recently completed, providing new insights into its biology and evolution. Moreover, new cell biology approaches and the use of novel inhibitors have allowed detailed investigations of its interaction with host cells.