The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

Inhibition of host cell apoptosis is crucial for survival and replication of several intracellular bacterial pathogens. To interfere with apoptotic pathways, some pathogens use specialized secretion systems to inject bacterial effector proteins into the host cell cytosol. One of these pathogens is the obligate intracellular bacterium Coxiella burnetii, the etiological agent of the zoonotic disease Q fever.

In this study, we analyzed the molecular activity of the anti-apoptotic T4SS effector protein AnkG (CBU0781) to understand how C. burnetii manipulates host cell viability. We demonstrate by co- and RNA-immunoprecipitation that AnkG binds to the host cell DExD box RNA helicase 21 (DDX21) as well as to the host cell 7SK small nuclear ribonucleoprotein (7SK snRNP) complex, an important regulator of the positive transcription elongation factor b (P-TEFb). The co-immunoprecipitation of AnkG with DDX21 is probably mediated by salt bridges and is independent of AnkG-7SK snRNP binding, and vice versa. It is known that DDX21 facilitates the release of P-TEFb from the 7SK snRNP complex.

Consistent with the documented function of released P-TEFb in RNA Pol II pause release, RNA sequencing experiments confirmed AnkG-mediated transcriptional reprogramming and showed that expression of genes involved in apoptosis, trafficking, and transcription are influenced by AnkG. Importantly, DDX21 and P-TEFb are both essential for AnkG-mediated inhibition of host cell apoptosis, emphasizing the significance of the interaction of AnkG with both, the DDX21 protein and the 7SK RNA. In line with a critical function of AnkG in pathogenesis, the AnkG deletion C. burnetii strain was severely affected in its ability to inhibit host cell apoptosis and to generate a replicative C. burnetii-containing vacuole. In conclusion, the interference with the activity of regulatory host cell RNAs mediated by a bacterial effector protein represent a novel mechanism through which C. burnetii modulates host cell transcription, thereby enhancing permissiveness to bacterial infection.

Source: PLoS Pathogens 18 (2022)

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Inactivation kinetics of Coxiella burnetii during high-temperature short-time pasteurization of milk:

 The Gram-negative, obligate intracellular bacterium Coxiella burnetii is the causative organism of the zoonosis Q fever and is known for its resistance toward various intra and extracellular stressors. Infected ruminants such as cattle, sheep, and goats can shed the pathogen in their milk. Pasteurization of raw milk was introduced for the inactivation of C. burnetii and other milk-borne pathogens. Legal regulations for the pasteurization of milk are mostly based on recommendations of the Codex Alimentarius. As described there, C. burnetii is considered as the most heat-resistant non-sporeforming bacterial pathogen in milk and has to be reduced by at least 5 log10-steps during the pasteurization process. However, the corresponding inactivation data for C. burnetii originate from experiments performed more than 60 years ago. Recent scientific findings and the technological progress of modern pasteurization equipment indicate that C. burnetii is potentially more effectively inactivated during pasteurization than demanded in the Codex Alimentarius. In the present study, ultra-high heat-treated milk was inoculated with different C. burnetii field isolates and subsequently heat-treated in a pilot-plant pasteurizer. Kinetic inactivation data in terms of D- and z-values were determined and used for the calculation of heat-dependent log reduction. With regard to the mandatory 5 log10-step reduction of the pathogen, the efficacy of the established heat treatment regime was confirmed, and, in addition, a reduction of the pasteurization temperature seems feasible.

Source: Marcel Wittwer, Philipp Hammer, Martin Runge, Peter Valentin-Weigand, Heinrich Neubauer, Klaus Henning und Katja Mertens-Scholz, Inactivation kinetics of Coxiella burnetii during high-temperature short-time pasteurization of milk
Frontiers in Microbiology, Vol. 12, 2022

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Cover story: A Q fever outbreak on a mixed dairy farm – a one health approach

 A Q fever outbreak on a dairy goat/cattle farm was investigated with regard to the One Health concept. Samples from all domestic animals and the farmer's family were collected, including goats with different reproductive statuses. The serological analysis revealed current infections in goats, whereas cows had ongoing infections. The possible influence of gestation and hormonal status on Coxiella burnetii infection in goats is discussed. A new cattle-associated Coxiella burnetii genotype C16 was identified in vaginal swabs and placentas from ruminants. Feline, canine, and human sera also tested positive for Coxiella burnetii antibodies. We conclude that the cattle herd may have transmitted Coxiella to the pregnant goats, resulting in zoonotic spread of Q fever to cats, dogs, and humans on the affected farm.

Source: Benjamin U. Bauer, Michael R. Knittler, T. Louise Herms, Dimitrios Frangoulidis, Svea Matthiesen, Dennis Tappe, Martin Runge and Martin Ganter
Cover story: A Q Fever Outbreak in a Mixed Dairy Farm - A one Health Approach
Publikation: Multispecies Q Fever Outbreak in a Mixed Dairy Goat and Cattle Farm Based on a New Bovine-Associated Genotype of Coxiella burnetii
Vet. Sci., Volume 8, Issue 11 (November 2021)

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Q fever outbreak at Charité in Berlin:

 According to a news story published by "Berliner Morgenpost" staff members were affected by a q fever outbreak at Charité in Mid-July 2021 after animal experiments with pregnant sheep. 20 staff members of the „Forschungseinrichtung für experimentelle Medizin" at the Virchow Clinique were infected with Coxiella burnetii when carrying out the animal experiments.

The trigger for the q fever specific diagnostics among staff members was obviously the detection of the pathogen in one of the experimental animals at the beginning of August 2021. At that time, it was possible to detect antibodies in staff members of the Charité, which indicated an acute q fever infection. Upon request by "Berliner Morgenpost" the Charité pointed out that all individuals who became ill have recovered in the meantime.

In addition, a Charité spokesman stated that the experimental animals came from breeding facilities in Brandenburg, where no q fever has been detected so far. Clinical signs have been absent in the sheep and there hasn't been any suspicion that the animals had been infected in the first place. Immediately after a first suspected infection within the Charité the holding of the research facilities was quarantined. Yet another consequence from the q fever outbreak is that stricter quality criteria regarding hygiene in the animal stock, supply chain and risk assessment will be paid attention to in future when purchasing animals, in order to reduce the risk of introducing zoonotic diseases. There hasn't been any obligation to check for the agent of q fever in farm animals regularly, so far.

Remarks:
Fortunately, institutions from the Q-GAPS research project (National Reference Laboratories for q fever, human and veterinary medicine, Lower State Office for Consumer Protection and Food Safety, Food and Veterinary Institute Braunschweig/Hannover, (LAVES)) also contributed to the analysis of the q fever outbreak described above. Based on the experience and knowledge of the Q-GAPS partners a guideline is being drawn up which contains information including recommendations for action concerning the overall topic q fever. The article about the outbreak in an establishment for animal use cited here shows an example of the necessity and importance of such a professional gathering of information and consulting. Therefore, it should be taken into account as to whether further protective measurements have to be taken or procedures for risk mitigation have to be considered, if necessary, not only in holdings of origin but also in research projects along these lines using experimental animals. For example, a regular q fever monitoring in as well as in experimental animals would be advisable, especially, as q fever infections can often be asymptomatic.

Image Sheep: © Stiftung Tierärztliche Hochschule Hannover

Sources:
Berliner Morgenpost: Tierseuchen-Ausbruch an der Charité (in German, for a fee)

BZ: Q-Fieber! 20 Charité-Mitarbeiter mit Tier-Seuche infiziert

Humoral immune response to Q fever vaccination of three sheep flocks naturally pre-infected with Coxiella burnetii:

 Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii; Coxiella-infected ruminants are the main reservoir shedding the pathogen during abortion or parturition through birth products. Germany has a long history of small-scale Q fever epidemics in the human population mostly associated with lambing sheep. Therefore, fast and efficient control measures are essentially required to prevent transmission from infected sheep flocks to humans.

In our present study, three sheep flocks were vaccinated with an inactivated C. burnetii phase I vaccine after a field infection with C. burnetii was diagnosed. Serum samples and vaginal swabs were collected at different time points to evaluate the extent of the outbreak and the consequences of the vaccination. The serum samples were examined by phase-specific IgG phase I and phase II ELISAs and a commercial ELISA, simultaneously detecting both phase variations. Moreover, vaginal swabs were analysed by qPCR. The fourth flock with no Q fever history and non-vaccinated animals were used as a control group to evaluate the phase-specific ELISAs.

The inactivated C. burnetii phase I vaccine induced an IgG phase II response and boosted the humoral immune reaction against natural preinfections. Furthermore, the longevity of vaccine-induced antibodies seems to depend on previous infections. Around 16 months after primary vaccination, mainly IgG phase I antibodies were detectable. Vaccination did not prevent shedding at the next lambing season. Most interestingly, the phasespecific ELISAs revealed more C. burnetii positive animals than the blended ELISA-Assay. Taken together, phase-specific ELISAs are suitable tools to provide insights into natural- or vaccine-induced humoral immune responses to C. burnetii in sheep.

Original Abstract, © 2021 Elsevier Ltd. All rights reserved.

Source: Benjamin U. Bauer, Michael R. Knittler, T. Louise Prüfer, Annika Wolf, Svea Matthiesen, Martin Runge, Martin Ganter (2021) Humoral immune response to Q fever vaccination of three sheep flocks naturally pre-infected with Coxiella burnetii. Vaccine, Volume 39, Issue 10, 5 March 2021, Pages 1499-1507, https://doi.org/10.1016/j.vaccine.2021.01.062

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Concept of an Active Surveillance System for Q Fever in German Small Ruminants - Conflicts Between Best Practices and Feasibility

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Inhalation of contaminated dust particles or aerosols originating from animals (esp. small ruminants) is the main source of human infection. Hence, an active early warning system for Q fever in German small ruminant livestock was conceptualized to prevent human infections.

First, we describe the best practice for establishing this system before evaluating its feasibility, as the combination of both evokes conflicts. Vaginal swabs from all husbandry systems with a focus on reproductive females should pooled and investigated by PCR to detect C. burnetii-shedding animals. Multistage risk-based sampling shall be carried out at the flock level and within-flock level. At the flock level, all flocks that are at risk to transmit the pathogen to the public must be sampled. At the within-flock level, all primi- and multiparous females after lambing must be tested in order to increase the probability of identifying a positive herd. Sampling should be performed during the main lambing period and before migration in residential areas.

Furthermore, individual animals should be tested before migration or exhibition to ensure a negative status. If a flock tests positive in at least one individual sample, then flock-specific preventive measures should be implemented. This approach implies huge financial costs (sample testing, action/control measures). Hence, taking the step to develop more feasible and affordable preventive measures, e.g., vaccinating small ruminant flocks, should replace testing wherever justifiable.

Abstract taken from: Winter F, Schoneberg C, Wolf A, Bauer BU, Prüfer TL, Fischer SF, Gerdes U, Runge M, Ganter M and Campe A (2021) Concept of an Active Surveillance System for Q Fever in German Small Ruminants — Conflicts Between Best Practices and Feasibility. Front. Vet. Sci. 8:623786. doi: 10.3389/fvets.2021.623786

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