Brucella Egresses from Host Cells Exploiting Multivesicular Bodies.

Host cell egress is a critical step in the life cycle of intracellular pathogens, especially in microbes capable of establishing chronic infections. The Gram-negative bacterium Brucella belongs to such a group of pathogens. Even though much has been done to understand how Brucella avoids killing and multiplies in its intracellular niche, the mechanism that this bacterium deploys to egress from the cell to complete its cycle has been poorly studied. In the manuscript, we quantify the kinetics of bacterial egress and show that Brucella exploits multivesicular bodies to exit host cells. For the first time, we visualized the process of egress in real time by live video microscopy and showed that a population of intracellular bacteria exit from host cells in vacuoles containing multivesicular body-like features. We observed the colocalization of Brucella with two multivesicular markers, namely, CD63 and LBPA, both during the final stages of the intracellular life cycle and in egressed bacteria. Moreover, drugs that either promote or inhibit multivesicular bodies either increased or decreased the number of extracellular bacteria, respectively. Our results strongly suggest that Brucella hijacks multivesicular bodies to exit the host cells to initiate new infection events. IMPORTANCE How intracellular bacterial pathogens egress from host cells has been poorly studied. This is particularly important because this stage of the infectious cycle can have a strong impact on how the host resolves the infection. Brucella is an intracellular pathogen that infects mammals, including humans, and causes a chronic debilitating illness. The bacterium has evolved a plethora of mechanisms to invade host cells, avoid degradation in the endocytic pathway, and actively multiply within a specialized intracellular compartment. However, how this pathogen exits from infected cells to produce reinfection and complete its life cycle is poorly understood. In the manuscript, we shed some light on the mechanisms that are exploited by Brucella to egress from host cells. We observed for the first time the egress of Brucella from infected cells by time-lapse video microscopy, and we found that the bacterium exits in vesicles containing multivesicular bodies (MVBs) features. Moreover, the drug manipulation of MVBs resulted in the alteration of bacterial egress efficiency. Our results indicate that Brucella hijacks MVBs to exit host cells and that this strongly contributes to the reinfection cycle.

VirJ Is a Brucella Virulence Factor Involved in the Secretion of Type IV Secreted Substrates.

The VirB secretion apparatus in Brucella belongs to the type IV secretion systems present in many pathogenic bacteria and is absolutely necessary for the efficient evasion of the Brucella-containing vacuole from the phagocytic route in professional phagocytes. This system is responsible for the secretion of a plethora of effector proteins that alter the biology of the host cell and promote the intracellular replication process. Although many VirB substrates have been identified in Brucella, we still know very little about the secretion mechanism that mediates their translocation across the two membranes and the periplasmic space. In this manuscript, we describe the identification of a gene, virJ, that codes for a protein with periplasmic localization that is involved in the intracellular replication process and virulence in mice. Our analysis revealed that this protein is necessary for the secretion of at least two VirB substrates that have a periplasmic intermediate and that it directly interacts with them. We additionally show that VirJ also associates with the apparatus per se and that its absence affects the assembly of the complex. We hypothesize that VirJ is part of a secretion platform composed of the translocon and several secretion substrates and that it probably coordinates the proper assembly of this macromolecular complex.

BigA is a novel adhesin of Brucella that mediates adhesion to epithelial cells.

Adhesion to cells is the initial step in the infectious cycle of basically all pathogenic bacteria, and to do so, microorganisms have evolved surface molecules that target different cellular receptors. Brucella is an intracellular pathogen that infects a wide range of mammals whose virulence is completely dependent on the capacity to replicate in phagocytes. Although much has been done to elucidate how Brucella multiplies in macrophages, we still do not understand how bacteria invade epithelial cells to perform a replicative cycle or what adhesion molecules are involved in the process. We report the identification in Brucella abortus of a novel adhesin that harbours a bacterial immunoglobulin-like domain and demonstrate that this protein is involved in the adhesion to polarized epithelial cells such as the Caco-2 and Madin-Darby canine kidney models targeting the bacteria to the cell-cell interaction membrane. While deletion of the gene significantly reduced adhesion, over-expression dramatically increased it. Addition of the recombinant protein to cells induced cytoskeleton rearrangements and showed that this adhesin targets proteins of the cell-cell interaction membrane in confluent cultures.

A B lymphocyte mitogen is a Brucella abortus virulence factor required for persistent infection.

Microbial pathogens with the ability to establish chronic infections have evolved strategies to actively modulate the host immune response. Brucellosis is a disease caused by a Gram-negative intracellular pathogen that if not treated during the initial phase of the infection becomes chronic as the bacteria persist for the lifespan of the host. How this pathogen and others achieve this action is a largely unanswered question. We report here the identification of a Brucella abortus gene (prpA) directly involved in the immune modulation of the host. PrpA belongs to the proline-racemase family and elicits a B lymphocyte polyclonal activation that depends on the integrity of its proline-racemase catalytic site. Stimulation of splenocytes with PrpA also results in IL-10 secretion. Construction of a B. abortus-prpA mutant allowed us to assess the contribution of PrpA to the infection process. Mice infected with B. abortus induced an early and transient nonresponsive status of splenocytes to both Escherichia coli LPS and ConA. This phenomenon was not observed when mice were infected with a B. abortus-prpA mutant. Moreover, the B. abortus-prpA mutant had a reduced capacity to establish a chronic infection in mice. We propose that an early and transient nonresponsive immune condition of the host mediated by this B cell polyclonal activator is required for establishing a successful chronic infection by Brucella.

Estudio del primer activador policlonal de linfocitos B en Brucella abortus y sus implicancias en inmunoevasión e inmunodepresión durante la etapa aguda de la infección

Conclusiones: Utilizando la cepa de Brucella abortus mutante para rac1 (B.abortus-rac1) hemos demostrado que el gen rac1 de Brucella abortus es requerido para la producción de un incremento en el número total de linfocitos T y B de bazo durante la etapa aguda de la infección. Además, este aumento policlonal de células B y T observado en ratones infectados produce un estado transiente de anergia o de falta de respuesta de dichos linfocitos T y B, que se refleja en una falta de respuesta frente a la estimulación con mitógenos convencionales como LPS o ConA, y que es revertido a las 11 semanas post infección. Estos fenómenos de linfoproliferación e inmunosupresión parecen ser requeridos por la bacteria para montar una infección crónica eficiente, ya que la persistencia de B. abortus-rac1 es 100 veces menor que la de su cepa parental en cronicidad. Los resultados obtenidos a lo largo de este trabajo en conjunto con aquellos obtenidos en experimentos anteriores demuestran claramente que rac1 es un nuevo factor de virulencia de Brucella abortus. I

Estudio del primer activador policlonal de linfocitos B en Brucella abortus y sus implicancias en inmunoevasión e inmunodepresión durante la etapa aguda de la infección

Conclusiones: Utilizando la cepa de Brucella abortus mutante para rac1 (B.abortus-rac1) hemos demostrado que el gen rac1 de Brucella abortus es requerido para la producción de un incremento en el número total de linfocitos T y B de bazo durante la etapa aguda de la infección. Además, este aumento policlonal de células B y T observado en ratones infectados produce un estado transiente de anergia o de falta de respuesta de dichos linfocitos T y B, que se refleja en una falta de respuesta frente a la estimulación con mitógenos convencionales como LPS o ConA, y que es revertido a las 11 semanas post infección. Estos fenómenos de linfoproliferación e inmunosupresión parecen ser requeridos por la bacteria para montar una infección crónica eficiente, ya que la persistencia de B. abortus-rac1 es 100 veces menor que la de su cepa parental en cronicidad. Los resultados obtenidos a lo largo de este trabajo en conjunto con aquellos obtenidos en experimentos anteriores demuestran claramente que rac1 es un nuevo factor de virulencia de Brucella abortus. I

Estudio del primer activador policlonal de linfocitos B en Brucella abortus y sus implicancias en inmunoevasión e inmunodepresión durante la etapa aguda de la infección

Conclusiones: Utilizando la cepa de Brucella abortus mutante para rac1 (B.abortus-rac1) hemos demostrado que el gen rac1 de Brucella abortus es requerido para la producción de un incremento en el número total de linfocitos T y B de bazo durante la etapa aguda de la infección. Además, este aumento policlonal de células B y T observado en ratones infectados produce un estado transiente de anergia o de falta de respuesta de dichos linfocitos T y B, que se refleja en una falta de respuesta frente a la estimulación con mitógenos convencionales como LPS o ConA, y que es revertido a las 11 semanas post infección. Estos fenómenos de linfoproliferación e inmunosupresión parecen ser requeridos por la bacteria para montar una infección crónica eficiente, ya que la persistencia de B. abortus-rac1 es 100 veces menor que la de su cepa parental en cronicidad. Los resultados obtenidos a lo largo de este trabajo en conjunto con aquellos obtenidos en experimentos anteriores demuestran claramente que rac1 es un nuevo factor de virulencia de Brucella abortus. I