In Vitro Antibacterial Activity of Microbial Natural Products against Bacterial Pathogens of Veterinary and Zoonotic Relevance.

Antimicrobial resistance (AMR) is considered one of the greatest threats to both human and animal health. Efforts to address AMR include implementing antimicrobial stewardship programs and introducing alternative treatment options. Nevertheless, effective treatment of infectious diseases caused by bacteria will still require the identification and development of new antimicrobial agents. Eight different natural products were tested for antimicrobial activity against seven pathogenic bacterial species (Brachyspira sp., Chlamydia sp., Clostridioides sp., Mannheimia sp., Mycobacterium sp., Mycoplasma sp., Pasteurella sp.). In a first pre-screening, most compounds (five out of eight) inhibited bacterial growth only at high concentrations, but three natural products (celastramycin A [CA], closthioamide [CT], maduranic acid [MA]) displayed activity at concentrations <2 µg/mL against Pasteurella sp. and two of them (CA and CT) also against Mannheimia sp. Those results were confirmed by testing a larger collection of isolates encompassing 64 Pasteurella and 56 Mannheimia field isolates originating from pigs or cattle, which yielded MIC90 values of 0.5, 0.5, and 2 µg/mL against Pasteurella and 0.5, 4, and >16 µg/mL against Mannheimia for CA, CT, and MA, respectively. CA, CT, and MA exhibited higher MIC50 and MIC90 values against Pasteurella isolates with a known AMR phenotype against commonly used therapeutic antimicrobial agents than against isolates with unknown AMR profiles. This study demonstrates the importance of whole-cell antibacterial screening of natural products to identify promising scaffolds with broad- or narrow-spectrum antimicrobial activity against important Gram-negative veterinary pathogens with zoonotic potential.

Interdisciplinary studies on Coxiella burnetii: From molecular to cellular, to host, to one health research.

The Q-GAPS (Q fever GermAn interdisciplinary Program for reSearch) consortium was launched in 2017 as a German consortium of more than 20 scientists with exceptional expertise, competence, and substantial knowledge in the field of the Q fever pathogen Coxiella (C.) burnetii. C. burnetii exemplifies as a zoonotic pathogen the challenges of zoonotic disease control and prophylaxis in human, animal, and environmental settings in a One Health approach. An interdisciplinary approach to studying the pathogen is essential to address unresolved questions about the epidemiology, immunology, pathogenesis, surveillance, and control of C. burnetii. In more than five years, Q-GAPS has provided new insights into pathogenicity and interaction with host defense mechanisms. The consortium has also investigated vaccine efficacy and application in animal reservoirs and identified expanded phenotypic and genotypic characteristics of C. burnetii and their epidemiological significance. In addition, conceptual principles for controlling, surveilling, and preventing zoonotic Q fever infections were developed and prepared for specific target groups. All findings have been continuously integrated into a Web-based, interactive, freely accessible knowledge and information platform (www.q-gaps.de), which also contains Q fever guidelines to support public health institutions in controlling and preventing Q fever. In this review, we will summarize our results and show an example of how an interdisciplinary consortium provides knowledge and better tools to control a zoonotic pathogen at the national level.

Genome-wide association reveals host-specific genomic traits in Escherichia coli.

BACKGROUND: Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. RESULTS: We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. CONCLUSIONS: This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

Bovine blood derived macrophages are unable to control Coxiella burnetii replication under hypoxic conditions.

Background: Coxiella burnetii is a zoonotic pathogen, infecting humans, livestock, pets, birds and ticks. Domestic ruminants such as cattle, sheep, and goats are the main reservoir and major cause of human infection. Infected ruminants are usually asymptomatic, while in humans infection can cause significant disease. Human and bovine macrophages differ in their permissiveness for C. burnetii strains from different host species and of various genotypes and their subsequent host cell response, but the underlying mechanism(s) at the cellular level are unknown. Methods: C. burnetii infected primary human and bovine macrophages under normoxic and hypoxic conditions were analyzed for (i) bacterial replication by CFU counts and immunofluorescence; (ii) immune regulators by westernblot and qRT-PCR; cytokines by ELISA; and metabolites by gas chromatography-mass spectrometry (GC-MS). Results: Here, we confirmed that peripheral blood-derived human macrophages prevent C. burnetii replication under oxygen-limiting conditions. In contrast, oxygen content had no influence on C. burnetii replication in bovine peripheral blood-derived macrophages. In hypoxic infected bovine macrophages, STAT3 is activated, even though HIF1α is stabilized, which otherwise prevents STAT3 activation in human macrophages. In addition, the TNFα mRNA level is higher in hypoxic than normoxic human macrophages, which correlates with increased secretion of TNFα and control of C. burnetii replication. In contrast, oxygen limitation does not impact TNFα mRNA levels in C. burnetii-infected bovine macrophages and secretion of TNFα is blocked. As TNFα is also involved in the control of C. burnetii replication in bovine macrophages, this cytokine is important for cell autonomous control and its absence is partially responsible for the ability of C. burnetii to replicate in hypoxic bovine macrophages. Further unveiling the molecular basis of macrophage-mediated control of C. burnetii replication might be the first step towards the development of host directed intervention measures to mitigate the health burden of this zoonotic agent.

Clostridioides difficile in South American Camelids in Germany: First Insights into Molecular and Genetic Characteristics and Antimicrobial Resistance.

Little is known about zoonotic pathogens and their antimicrobial resistance in South American camelids (SAC) in Germany including Clostridioides (C.) difficile. The aim of this study was to investigate prevalence, molecular characteristics and antimicrobial resistance of C. difficile in SAC. Composite SAC faecal samples were collected in 43 husbandries in Central Germany and cultured for C. difficile. Toxinotyping and ribotyping was done by PCR. Whole genome sequencing was performed with Illumina® Miseq™. The genomes were screened for antimicrobial resistance determinants. Genetic relatedness of the isolates was investigated using core genome multi locus sequence typing (cgMLST) and single nucleotide polymorphism analysis. Antimicrobial susceptibility testing was done using the Etest® method. Eight C. difficile isolates were recovered from seven farms. The isolates belonged to different PCR ribotypes. All isolates were toxinogenic. cgMLST revealed a cluster containing isolates recovered from different farms. Seven isolates showed similar resistance gene patterns. Different phenotypic resistance patterns were found. Agreement between phenotypic and genotypic resistance was identified only in some cases. Consequently, SAC may act as a reservoir for C. difficile. Thus, SAC may pose a risk regarding zoonotic transmission of toxinogenic, potentially human-pathogenic and resistant C. difficile isolates.

Portable Differential Detection of CTX-M ESBL Gene Variants, blaCTX-M-1 and blaCTX-M-15, from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification.

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamase (ESBL) enzymes produced by Enterobacteriaceae confer resistance to clinically relevant third-generation cephalosporins. CTX-M group 1 variants, CTX-M-1 and CTX-M-15, are the leading ESBL-producing Enterobacteriaceae associated with animal and human infection, respectively, and are an increasing antimicrobial resistance (AMR) global health concern. The blaCTX-M-1 and blaCTX-M-15 genes encoding these variants have an approximate nucleotide sequence similarity of 98.7%, making effective differential diagnostic monitoring difficult. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) enables rapid real-time multiplex pathogen detection with single-base specificity and portable on-site testing. We have developed an internally controlled multiplex CTX-M-1/15 LEC-LAMP assay for the differential detection of blaCTX-M-1 and blaCTX-M-15. Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for blaCTX-M-1 (n = 18), blaCTX-M-15 (n = 35), and other closely related blaCTX-Ms (n = 38) from Ireland, Germany, and Portugal, with analytical sensitivity determined using probit regression analysis. Animal fecal sample testing using the CTX-M-1/15 LEC-LAMP assay in combination with a rapid DNA extraction protocol was carried out on porcine fecal samples previously confirmed to be PCR-positive for E. coli blaCTX-M. Portable instrumentation was used to further analyze each fecal sample and demonstrate the on-site testing capabilities of the LEC-LAMP assay with the rapid DNA extraction protocol. The CTX-M-1/15 LEC-LAMP assay demonstrated complete analytical specificity for the differential detection of both variants with sensitive low-level detection of 8.5 and 9.8 copies per reaction for blaCTX-M-1 and blaCTX-M-15, respectively, and E. coli blaCTX-M-1 was identified in all blaCTX-M positive porcine fecal samples tested. IMPORTANCE CTX-M ESBL-producing E. coli is an increasing AMR public health issue with the transmission between animals and humans via zoonotic pathogens now a major area of interest. Accurate and timely identification of ESBL-expressing E. coli CTX-M variants is essential for disease monitoring, targeted antibiotic treatment and infection control. This study details the first report of portable diagnostics technology for the rapid differential detection of CTX-M AMR markers blaCTX-M-1 and blaCTX-M-15, facilitating improved identification and surveillance of these closely related variants. Further application of this portable internally controlled multiplex CTX-M-1/15 LEC-LAMP assay will provide new information on the transmission and prevalence of these CTX-M ESBL alleles. Furthermore, this transferable diagnostic technology can be applied to other new and emerging relevant AMR markers of interest providing more efficient and specific portable pathogen detection for improved epidemiological surveillance.

Comparative Genomic Analysis of Antimicrobial-Resistant Escherichia coli from South American Camelids in Central Germany.

South American camelids (SAC) are increasingly kept in Europe in close contact with humans and other livestock species and can potentially contribute to transmission chains of epizootic, zoonotic and antimicrobial-resistant (AMR) agents from and to livestock and humans. Consequently, SAC were included as livestock species in the new European Animal Health Law. However, the knowledge on bacteria exhibiting AMR in SAC is too scarce to draft appropriate monitoring and preventive programs. During a survey of SAC holdings in central Germany, 39 Escherichia coli strains were isolated from composite fecal samples by selecting for cephalosporin or fluoroquinolone resistance and were here subjected to whole-genome sequencing. The data were bioinformatically analyzed for strain phylogeny, detection of pathovars, AMR genes and plasmids. Most (33/39) strains belonged to phylogroups A and B1. Still, the isolates were highly diverse, as evidenced by 28 multi-locus sequence types. More than half of the isolates (23/39) were genotypically classified as multidrug resistant. Genes mediating resistance to trimethoprim/sulfonamides (22/39), aminoglycosides (20/39) and tetracyclines (18/39) were frequent. The most common extended-spectrum-ß-lactamase gene was blaCTX-M-1 (16/39). One strain was classified as enteropathogenic E. coli. The positive results indicate the need to include AMR bacteria in yet-to-be-established animal disease surveillance protocols for SAC.

Using unique ORFan genes as strain-specific identifiers for Escherichia coli.

BACKGROUND: Bacterial identification at the strain level is a much-needed, but arduous and challenging task. This study aimed to develop a method for identifying and differentiating individual strains among multiple strains of the same bacterial species. The set used for testing the method consisted of 17 Escherichia coli strains picked from a collection of strains isolated in Germany, Spain, the United Kingdom and Vietnam from humans, cattle, swine, wild boars, and chickens. We targeted unique or rare ORFan genes to address the problem of selective and specific strain identification. These ORFan genes, exclusive to each strain, served as templates for developing strain-specific primers. RESULTS: Most of the experimental strains (14 out of 17) possessed unique ORFan genes that were used to develop strain-specific primers. The remaining three strains were identified by combining a PCR for a rare gene with a selection step for isolating the experimental strains. Multiplex PCR allowed the successful identification of the strains both in vitro in spiked faecal material in addition to in vivo after experimental infections of pigs and recovery of bacteria from faecal material. In addition, primers for qPCR were also developed and quantitative readout from faecal samples after experimental infection was also possible. CONCLUSIONS: The method described in this manuscript using strain-specific unique genes to identify single strains in a mixture of strains proved itself efficient and reliable in detecting and following individual strains both in vitro and in vivo, representing a fast and inexpensive alternative to more costly methods.

Looking Inside Non-Destructively: Label-Free, Raman-Based Visualization of Intracellular Coxiella burnetii.

The life cycle of intracellular pathogens is often complex and can include different morphoforms. Treatment of intracellular infections and unperturbed studying of the pathogen inside the host cell are frequently challenging. Here, we present a Raman-based, label-free, non-invasive, and non-destructive method to localize, visualize, and even quantify intracellular bacteria in 3D within intact host cells in a Coxiella burnetii infection model. C. burnetii is a zoonotic obligate intracellular pathogen that causes infections in ruminant livestock and humans with an acute disease known as Q fever. Using statistical data analysis, no isolation is necessary to gain detailed information on the intracellular pathogen's metabolic state. High-quality false color image stacks with diffraction-limited spatial resolution enable a 3D spatially resolved single host cell analysis that shows excellent agreement with results from transmission electron microscopy. Quantitative analysis at different time points post infection allows to follow the infection cycle with the transition from the large cell variant (LCV) to the small cell variant (SCV) at around day 6 and a gradual change in the lipid composition during vacuole maturation. Spectral characteristics of intracellular LCV and SCV reveal a higher lipid content of the metabolically active LCV.

Interaction of Salmonella Gallinarum and Salmonella Enteritidis with peripheral leucocytes of hens with different laying performance.

Salmonella enterica ssp. enterica serovars Enteritidis (SE) and Gallinarum (SG) cause different diseases in chickens. However, both are able to reach the blood stream where heterophils and monocytes are potentially able to phagocytose and kill the pathogens. Using an ex vivo chicken whole blood infection model, we compared the complex interactions of the differentially host-adapted SE and SG with immune cells in blood samples of two White Leghorn chicken lines showing different laying performance (WLA: high producer; R11: low producer). In order to examine the dynamic interaction between peripheral blood leucocytes and the Salmonella serovars, we performed flow cytometric analyses and survival assays measuring (i) leucocyte numbers, (ii) pathogen association with immune cells, (iii) Salmonella viability and (iv) immune gene transcription in infected whole blood over a four-hour co-culture period. Inoculation of blood from the two chicken lines with Salmonella led primarily to an interaction of the bacteria with monocytes, followed by heterophils and thrombocytes. We found higher proportions of monocytes associated with SE than with SG. In blood samples of high producing chickens, a decrease in the numbers of both heterophils and Salmonella was observed. The Salmonella challenge induced transcription of interleukin-8 (IL-8) which was more pronounced in SG- than SE-inoculated blood of R11. In conclusion, the stronger interaction of monocytes with SE than SG and the better survivability of Salmonella in blood of low-producer chickens shows that the host-pathogen interaction and the strength of the immune defence depend on both the Salmonella serovar and the chicken line.

Newly Isolated Animal Pathogen Corynebacterium silvaticum Is Cytotoxic to Human Epithelial Cells.

Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.

Shiga Toxin-Producing E. coli in Animals: Detection, Characterization, and Virulence Assessment.

Cattle and other ruminants are primary reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains which have a highly variable, but unpredictable, pathogenic potential for humans. Domestic swine can carry and shed STEC, but only STEC strains producing the Shiga toxin (Stx) 2e variant and causing edema disease in piglets are considered pathogens of veterinary medical interest. In this chapter, we present general diagnostic workflows for sampling livestock animals to assess STEC prevalence, magnitude, and duration of host colonization. This is followed by detailed method protocols for STEC detection and typing at genetic and phenotypic levels to assess the relative virulence exerted by the strains.

The Coxiella burnetii T4SS Effector AnkF Is Important for Intracellular Replication.

Coxiella burnetii is an obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Following uptake by alveolar macrophages, the pathogen replicates in an acidic phagolysosomal vacuole, the C. burnetii-containing vacuole (CCV). Effector proteins translocated into the host cell by the type IV secretion system (T4SS) are important for the establishment of the CCV. Here we focus on the effector protein AnkF and its role in establishing the CCV. The C. burnetii AnkF knock out mutant invades host cells as efficiently as wild-type C. burnetii, but this mutant is hampered in its ability to replicate intracellularly, indicating that AnkF might be involved in the development of a replicative CCV. To unravel the underlying reason(s), we searched for AnkF interactors in host cells and identified vimentin through a yeast two-hybrid approach. While AnkF does not alter vimentin expression at the mRNA or protein levels, the presence of AnkF results in structural reorganization and vesicular co-localization with recombinant vimentin. Ectopically expressed AnkF partially accumulates around the established CCV and endogenous vimentin is recruited to the CCV in a time-dependent manner, suggesting that AnkF might attract vimentin to the CCV. However, knocking-down endogenous vimentin does not affect intracellular replication of C. burnetii. Other cytoskeletal components are recruited to the CCV and might compensate for the lack of vimentin. Taken together, AnkF is essential for the establishment of the replicative CCV, however, its mode of action is still elusive.

The anti-apoptotic Coxiella burnetii effector protein AnkG is a strain specific virulence factor.

The ability to inhibit host cell apoptosis is important for the intracellular replication of the obligate intracellular pathogen Coxiella burnetii, as it allows the completion of the lengthy bacterial replication cycle. Effector proteins injected into the host cell by the C. burnetii type IVB secretion system (T4BSS) are required for the inhibition of host cell apoptosis. AnkG is one of these anti-apoptotic effector proteins. The inhibitory effect of AnkG requires its nuclear localization, which depends on p32-dependent intracellular trafficking and importin-α1-mediated nuclear entry of AnkG. Here, we compared the sequences of ankG from 37 C. burnetii isolates and classified them in three groups based on the predicted protein size. The comparison of the three different groups allowed us to identify the first 28 amino acids as essential and sufficient for the anti-apoptotic activity of AnkG. Importantly, only the full-length protein from the first group is a bona fide effector protein injected into host cells during infection and has anti-apoptotic activity. Finally, using the Galleria mellonella infection model, we observed that AnkG from the first group has the ability to attenuate pathology during in vivo infection, as it allows survival of the larvae despite bacterial replication.

Metabolic Traits of Bovine Shiga Toxin-Producing Escherichia Coli (STEC) Strains with Different Colonization Properties.

Cattle harbor Shiga toxin-producing Escherichia coli (STEC) in their intestinal tract, thereby providing these microorganisms with an ecological niche, but without this colonization leading to any clinical signs. In a preceding study, genotypic characterization of bovine STEC isolates unveiled that their ability to colonize cattle persistently (STECper) or only sporadically (STECspo) is more closely associated with the overall composition of the accessory rather than the core genome. However, the colonization pattern could not be unequivocally linked to the possession of classical virulence genes. This study aimed at assessing, therefore, if the presence of certain phenotypic traits in the strains determines their colonization pattern and if these can be traced back to distinctive genetic features. STECspo strains produced significantly more biofilm than STECper when incubated at lower temperatures. Key substrates, the metabolism of which showed a significant association with colonization type, were glyoxylic acid and L-rhamnose, which were utilized by STECspo, but not or only by some STECper. Genomic sequences of the respective glc and rha operons contained mutations and frameshifts in uptake and/or regulatory genes, particularly in STECper. These findings suggest that STECspo conserved features leveraging survival in the environment, whereas the acquisition of a persistent colonization phenotype in the cattle reservoir was accompanied by the loss of metabolic properties and genomic mutations in the underlying genetic pathways.

Dynamic Interplay of Host and Pathogens in an Avian Whole-Blood Model.

Microbial survival in blood is an essential step toward the development of disseminated diseases and blood stream infections. For poultry, however, little is known about the interactions of host cells and pathogens in blood. We established an ex vivo chicken whole-blood infection assay as a tool to analyze interactions between host cells and three model pathogens, Escherichia coli, Staphylococcus aureus, and Candida albicans. Following a systems biology approach, we complemented the experimental measurements with functional and quantitative immune characteristics by virtual infection modeling. All three pathogens were killed in whole blood, but each to a different extent and with different kinetics. Monocytes, and to a lesser extent heterophils, associated with pathogens. Both association with host cells and transcriptional activation of genes encoding immune-associated functions differed depending on both the pathogen and the genetic background of the chickens. Our results provide first insights into quantitative interactions of three model pathogens with different immune cell populations in avian blood, demonstrating a broad spectrum of different characteristics during the immune response that depends on the pathogen and the chicken line.

Microarray-based detection of resistance and virulence factors in commensal Escherichia coli from livestock and farmers in Egypt.

The objective of our study was to provide a molecular analysis using DNA-microarray based assays of commensal E. coli populations from apparently healthy livestock and their attendants to assess the virulence potential as well as multidrug resistance (MDR) genotypes. We randomly collected 132 fecal samples from seemingly healthy smallholder´s food producing animals [buffalo (n = 32) and cattle (n = 50)] as well as from contacting farmers (n = 50). Bacterial isolation and identification were performed using standard protocols, while E. coli isolates were characterized using a DNA microarray system targeting 60 different virulence and 47 antibiotic resistance genes of clinical importance and allowing assignment to most common H and O types. From the fecal samples examined, 47 E. coli isolates were obtained. The array predicted serotypes for 14 out of the 47 E. coli isolates. Six E. coli isolates were identified as STEC since Shiga toxin genes were detected. In summary, 36 different virulence genes were identified; of which, hemL, lpfA and iss were most prevalent. Thirty-four E. coli isolates were found to carry at least one antimicrobial resistance gene. Of these, 20 did exhibit genes allowing strain classification as MDR. More than half of the isolates contained antimicrobial resistance genes associated with beta lactam resistance 27/47 (57.5 %). The 13 remaining isolates did not contain any resistance gene tested with the array. Our study demonstrated the presence of antimicrobial resistance genes and virulence genotypes among commensal E. coli of human and animal sources.

Defying Death - How Coxiella burnetii Copes with Intentional Host Cell Suicide.

The obligate intracellular pathogen Coxiella burnetii is the causative agent of the worldwide zoonotic disease Q fever. This Gram-negative bacterium infects macrophages where it establishes a replicative niche in an acidic and phagolysosome-like vacuole. Establishing and maintaining the niche requires a functional type IV secretion system (T4SS) which translocates multiple effector proteins into the host cell. These effector proteins act by manipulating diverse cellular processes allowing the bacterium to establish an infection and complete its complex biphasic developmental cycle. The lengthy nature of this life cycle suggests that C. burnetii has to successfully deal with cellular defense processes. Cell death is one mechanism infected cells frequently utilize to control or to at least minimize the impact of an infection. To date, four effector proteins have been identified in C. burnetii, which interfere with the induction of cell death. Three, AnkG, CaeA, and CaeB, affect intrinsic apoptosis, CaeA additionally extrinsic apoptosis. The proteins target different steps of the apoptotic pathway and are not conserved among isolates suggesting redundancy as an important feature of cell death inhibition. The fourth effector protein, IcaA, interferes with the non-canonical pathway of pyroptosis, an important inflammatory cell death pathway for controlling infectious disease. Autophagy is relevant for the C. burnetii life-cycle, but to which extent autophagic cell death is a factor in bacterial survival and proliferation is still not clear. To convincingly understand how bacterial manipulation of autophagy affects cell death either directly or indirectly will require further experiments. Collectively, C. burnetii modulates the extrinsic and intrinsic apoptotic pathways and non-canonical pyroptosis to inhibit host cell death, thereby providing a stable, intracellular niche for the course of the pathogen's infectious cycle.

Modulation of host cell metabolism by T4SS-encoding intracellular pathogens.

Intracellular bacterial pathogens intimately interact with the infected host cell to prevent elimination and to ensure survival. One group of intracellular pathogens, including Coxiella burnetii, Legionella pneumophila, Brucella spp., Anaplasma phagocytophilum, and Ehrlichia chaffeensis, utilizes a type IV secretion system (T4SS) that injects effectors to modulate host cell signalling, vesicular trafficking, autophagy, cell death and transcription to ensure survival [1]. So far, little emphasis has been directed towards understanding how these bacteria manipulate host cell metabolism. This manipulation is not only important for gaining access to nutrients, but also for regulating specific virulence programs [2,3]. Here, we will summarize recent progress made in characterizing the manipulation of host cell metabolism by C. burnetii and other intracellular pathogens utilizing a T4SS.

Spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle as revealed by a genetic-insulators-containing dual-fluorescence HIV-1-based vector.

Long-lived latently HIV-1-infected cells represent a barrier to cure. We developed a dual-fluorescence HIV-1-based vector containing a pair of genetic insulators flanking a constitutive fluorescent reporter gene to study HIV-1 latency. The protective effects of these genetic insulators are demonstrated through long-term (up to 394 days) stable fluorescence profiles in transduced SUP-T1 cells. Analysis of 1,941 vector integration sites confirmed reproduction of HIV-1 integration patterns. We sorted monoclonal cells representing latent HIV-1 infections and found that both vector integration sites and integrity of the vector genomes influence the reactivation potentials of latent HIV-1 promoters. Interestingly, some latent monoclonal cells exhibited a small cell subpopulation with a spontaneously reactivated HIV-1 promoter. Higher expression levels of genes involved in cell cycle progression are observed in these cell subpopulations compared to their counterparts with HIV-1 promoters that remained latent. Consistently, larger fractions of spontaneously reactivated cells are in the S and G2 phases of the cell cycle. Furthermore, genistein and nocodazole treatments of these cell clones, which halted cells in the G2 phase, resulted in a 1.4-2.9-fold increase in spontaneous reactivation. Taken together, our HIV-1 latency model reveals that the spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle.