Ketterer, M. | Search bioRxiv

bioRxiv2024

The putative Type 4 secretion system effector BspD is involved in maintaining envelope integrity of the pathogen Brucella

Brucellosis is a debilitating disease caused by the Gram-negative, facultative intracellular zoonotic pathogen Brucella. En route to its intracellular replicative niche, Brucella encounters various stressful environments that may compromise envelop integrity. Here we show, that the proposed Type 4 secretion system (T4SS) effector BspD is a conserved protein of the Rhizobiales, which does not show signs of co-evolution with the presence of a T4SS or a certain lifestyle. We further present data indicating that BspD is critical for Brucella abortus envelope integrity in the stationary phase and in the presence of EDTA, a compound known to destabilize the outer membrane. Deletion of bspD resulted in abnormal bacterial morphologies, indicating its involvement in maintaining envelope integrity. Additionally, the absence of BspD led to the formation of fewer and smaller intracellular microcolonies in a macrophage infection model. From our observations, we propose that BspD of B. abortus is critical for preserving the integrity of the bacterial envelope, particularly under stressful conditions, which may enhance Brucellas ability to survive within host cells. ImportanceBrucellosis, caused by the intracellular pathogen Brucella, poses a significant health threat. Understanding how Brucella adapts to stressful environments is crucial. This study unveils BspD, a conserved protein within the Rhizobiales order, as a key player in maintaining Brucellas envelope integrity. Remarkably, BspDs function appears independent of the presence of a Type 4 Secretion System (T4SS) or specific lifestyle. Deletion of bspD resulted in compromised envelope integrity, abnormal bacterial morphologies, and reduced intracellular microcolony formation. These findings underscore BspDs critical role, particularly in stressful conditions like the stationary phase and EDTA exposure, and highlight its significance in Brucellas survival within host cells. This elucidation deepens our understanding of Brucella pathogenesis and may inform future therapeutic strategies against brucellosis.

microbiology↗

bioRxiv2022

Antibiotic persistence of Brucella abortus in its protective intracellular niche

BackgroundHuman Brucellosis caused by the facultative intracellular pathogen Brucella spp. is an endemic bacterial zoonosis manifesting as acute or chronic infections associated with high morbidity. Treatment typically involves a combination therapy of two antibiotics administered for several weeks to month, but despite this harsh treatment relapses occur at a rate of 5-15%. Although poor compliance and reinfection may account for a fraction of the observed relapse cases, it is apparent that the properties of the infectious agent itself may play a decisive role in this phenomenon. Methodology/Principal findingsWe used B. abortus carrying a dual reporter in a macrophage infection model to gain a better understanding of the efficacy of recommended therapies in cellulo. For this we used automated fluorescent microscopy as a prime read-out and developed specific CellProfiler pipelines to score infected macrophages at the population and the single cell level. Combining microscopy of constitutive and induced reporters with classical CFU determination, we quantified the protective nature of the Brucella intracellular niche to various antibiotics and the ability of B. abortus to persist in cellulo despite harsh antibiotic treatments. Conclusion/SignificanceWe demonstrate that treatment of infected macrophages with antibiotics at recommended concentrations fails to fully prevent growth and persistence of B. abortus in cellulo, which may be explained by a protective nature of the intracellular niche. Moreover, we show the presence of bona fide intracellular persisters upon antibiotic treatment, which are metabolically active and retain the full infectious potential, therefore constituting a plausible reservoir for reinfection and relapse. In conclusion, our results highlight the need to extend the spectrum of models to test new antimicrobial therapies for Brucellosis to better reflect the in vivo infection environment, and to develop therapeutic approaches targeting the persister subpopulation. Authors SummaryBrucellosis is a zoonosis endemic to many low- and middle-income countries around the world. Recommended therapies by the WHO are comprised of at least two antibiotics for several weeks, sometimes month. Despite these harsh treatments relapses are frequent. The underlying reasons for these relapses, besides reinfection and non-compliance to treatment, are unknown. Our study shows that Brucella abortus can form so called "persisters" under recommended treatments in rich broth but also inside macrophages. This small bacterial subpopulation survives antibiotic treatment and resumes growth after removal of the antibiotics and could therefore serve as a reservoir for relapses in human Brucellosis. Furthermore, we show that the macrophage intracellular niche of Brucella has protective properties against recommended antibiotics as observed for other intracellular pathogens, highlighting the necessity to develop new infection models to assess antibiotic efficacy.

microbiology↗