Biology subjects

Altamirano-Silva, P.

Publications and source records attributed to Altamirano-Silva, P..

4 recordsLinked to original sources

bioRxiv2025

Cell-to-cell transmission of Brucella abortus

Brucella abortus is a persistent intracellular pathogen capable of evading early immune responses. To explore the mechanism of exit of this bacterium, we monitored Brucella infection by live cell imaging for up to 100 hours. This approach uncovered various modes of bacterial spreading, including individual organisms, bacteria-containing vacuoles, and direct transmission between cells (called cell-to-cell transmission), each fueling new infections. This dissemination occurred regardless of antibiotic presence in the extracellular milieu. While receptor blockade using heparin suppressed free bacterial infections, it promoted cell-to-cell transmission, dependent on cell density. Cells participating in intercellular transmission survived longer than their non-infected counterparts. These observations underscore the adaptability of Brucella for propagating across diverse conditions. ImportanceThe intracellular life cycle of pathogenic bacteria encompasses adhesion, invasion, intracellular replication, and eventual exit from host cells. The exit phase is a critical step for pathogen persistence and dissemination to new hosts. Despite its relevance, the egress mechanisms of Brucella spp. remain largely unexplored. In this study, we describe three distinct modes of Brucella abortus exit: individual bacteria, bacterial clumps, and through direct cell-to-cell transmission. The latter may facilitate immune evasion by bypassing extracellular exposure, thereby contributing to the stealthy survival strategy of Brucella. The presence of multiple egress mechanisms suggests that B. abortus tailors its exit strategies according to its intracellular biogenesis and environmental cues.These findings not only advance our understanding of Brucella pathogenesis but also highlight potential avenues for targeting bacterial dissemination and persistence.

microbiology↗

bioRxiv2025

Brucella abortus egresses from host cells in infective clumps through an actin-dependent mechanism

Brucella abortus is an intracellular pathogen whose cell cycle encompasses attachment, internalization, trafficking, replication, and egress from cells. While the intracellular life of Brucella has been extensively studied, the mechanisms underlying its exit from host cells are still unclear. In this work, we observed a significant increase in the formation of B. abortus containing vacuoles intracellular clumps (BCVIC) and abundant B. abortus containing vacuoles-derived extracellular clumps (BCVEC) after 72 h of infection. Membrane extensions protruding through the polymerization of actin filaments were evident in cells at later stages of infection. Purified BCVIC and BCVEC were of similar sizes and predominantly acidic. These vacuoles exhibited a compact arrangement of well-ordered bacteria, comprised of a heterogeneous population of dead and live bacteria and host components LAMP-1 and actin filaments. A proportion of BCVEC were naked, while others were enclosed within an impermeable host membrane. The actin cytoskeleton was implicated in the protrusion of BCVEC since the modulation of Rho GTPases affected the egress of BCVEC. The BCVEC protruding from cells were capable of invading non-infected cells, initiating a new cycle of infection. The topological structure and function of BCVEC underscore their significance in the Brucella life cycle as vehicles for bacterial dissemination to host cells and organs. ImportanceB. abortus is an intracellular pathogen that traffics to the endoplasmic reticulum, where it replicates. However, the mechanisms by which Brucella exits host cells and infects new ones are not fully understood. We found that protruding acidic autophagic-like vesicles containing compact, well-organized Brucella clusters are shed from cells via GTPase and actin filament recruitment. Through this process, the vesicles are impermeable to antibodies and other substances, like antibiotics, and are highly infectious to neighboring cells. This previously unreported mechanism enhances understanding of the well-known Brucella stealth strategy to evade the immune system and establish harmful, long-lasting infections.

microbiology↗

bioRxiv2024

Clostridioides difficile binary toxin CDT induces biofilm-like persisting microcolonies

Clinical symptoms of Clostridioides difficile infection (CDI) range from diarrhea to pseudomembranous colitis. A major challenge in managing CDI is the high rate of relapse. Several studies correlate production of CDT binary toxin by clinical strains of Clostridioides difficile with higher relapse rates. Although the mechanism of action of CDT on host cells is known, its exact contribution to CDI is still unclear. To understand the physiological role of CDT during CDI, we established two hypoxic relevant intestinal models, Transwell and Microfluidic Intestine-on-Chip systems. Both were challenged with the epidemic strain UK1 CDT+ and its isogenic CDT- mutant. We report that CDT binary toxin induces mucin-associated microcolonies that increase C. difficile colonization and display biofilm-like properties by enhancing C. difficile resistance to vancomycin but not to fidaxomicin, a biofilm disrupting antibiotic. Importantly, biofilm- like CDT-dependent microcolonies were also observed in the caecum and colon of infected mice. Hence, our study shows that CDT toxin induces biofilm-like microcolonies, increasing C. difficile colonization and persistence.

microbiology↗

bioRxiv2021

c-di-AMP signaling is required for bile salts resistance and long-term colonization by Clostridioides difficile

To cause disease, the important human enteropathogen Clostridioides difficile must colonize the gastro-intestinal tract but little is known on how this organism senses and responds to the harsh host environment to adapt and multiply. Nucleotide second messengers are signaling molecules used by bacteria to respond to changing environmental conditions. In this study, we showed for the first time that c-di-AMP is produced by C. difficile and controls the uptake of potassium, making it essential for growth. We found that c-di-AMP is involved in biofilm formation, cell wall homeostasis, osmotolerance as well as detergent and bile salt resistance in C. difficile. In a colonization mouse model, a strain lacking GdpP, a c-di-AMP degrading enzyme, failed to persist in the gut in contrast to the parental strain. We identified OpuR as a new regulator that binds c-di-AMP and represses the expression of the compatible solute transporter OpuC. Interestingly, an opuR mutant is highly resistant to a hyperosmotic or bile salt stress compared to the parental strain while an opuCA mutant is more susceptible A short exposure of C. difficile cells to bile salts resulted in a decrease of the c-di-AMP concentrations reinforcing the hypothesis that changes in membrane characteristics due to variations of the cellular turgor or membrane damages constitute a signal for the adjustment of the intracellular c-di-AMP concentration. Thus, c-di-AMP is a signaling molecule with pleiotropic effects that controls osmolyte uptake to confer osmotolerance and bile salt resistance in C. difficile and that is important for colonization of the host. One Sentence Summaryc-di-AMP is an essential regulatory molecule conferring resistance to osmotic and bile salt stresses by controlling osmolyte uptake and contributing to gut persistence in the human enteropathogen Clostridioides difficile.

microbiology↗