Bordetella bronchiseptica diguanylate cyclase BdcB inhibits the type three secretion system and impacts the immune response.

Bordetella bronchiseptica is a gram-negative bacterium that causes respiratory diseases in different animals, including mice, making B. bronchiseptica the gold-standard model to investigate host-pathogen interaction at the molecular level. B. bronchiseptica utilizes many different mechanisms to precisely regulate the expression of virulence factors. Cyclic di-GMP is a second messenger synthesized by diguanylate cyclases and degraded by phosphodiesterases that regulates the expression of multiple virulence factors including biofilm formation. As in other bacteria, we have previously shown that c-di-GMP regulates motility and biofilm formation in B. bronchiseptica. This work describes the diguanylate cyclase BdcB (Bordetella diguanylate cyclase B) as an active diguanylate cyclase that promotes biofilm formation and inhibits motility in B. bronchiseptica. The absence of BdcB increased macrophage cytotoxicity in vitro and induced a greater production of TNF-α, IL-6, and IL-10 by macrophages. Our study reveals that BdcB regulates the expression of components of T3SS, an important virulence factor of B. bronchiseptica. The Bb∆bdcB mutant presented increased expression of T3SS-mediated toxins such as bteA, responsible for cytotoxicity. Our in vivo results revealed that albeit the absence of bdcB did not affect the ability of B. bronchiseptica to infect and colonize the respiratory tract of mice, mice infected with Bb∆bdcB presented a significantly higher pro-inflammatory response than those infected with wild type B. bronchiseptica.

Active Surveillance of Asymptomatic, Presymptomatic, and Oligosymptomatic SARS-CoV-2-Infected Individuals in Communities Inhabiting Closed or Semi-closed Institutions.

Background: The high COVID-19 dissemination rate demands active surveillance to identify asymptomatic, presymptomatic, and oligosymptomatic (APO) SARS-CoV-2-infected individuals. This is of special importance in communities inhabiting closed or semi-closed institutions such as residential care homes, prisons, neuropsychiatric hospitals, etc., where risk people are in close contact. Thus, a pooling approach-where samples are mixed and tested as single pools-is an attractive strategy to rapidly detect APO-infected in these epidemiological scenarios. Materials and Methods: This study was done at different pandemic periods between May 28 and August 31 2020 in 153 closed or semi-closed institutions in the Province of Buenos Aires (Argentina). We setup pooling strategy in two stages: first a pool-testing followed by selective individual-testing according to pool results. Samples included in negative pools were presumed as negative, while samples from positive pools were re-tested individually for positives identification. Results: Sensitivity in 5-sample or 10-sample pools was adequate since only 2 Ct values were increased with regard to single tests on average. Concordance between 5-sample or 10-sample pools and individual-testing was 100% in the Ct ≤ 36. We tested 4,936 APO clinical samples in 822 pools, requiring 86-50% fewer tests in low-to-moderate prevalence settings compared to individual testing. Conclusions: By this strategy we detected three COVID-19 outbreaks at early stages in these institutions, helping to their containment and increasing the likelihood of saving lives in such places where risk groups are concentrated.

Bordetella bronchiseptica Diguanylate Cyclase BdcA Regulates Motility and Is Important for the Establishment of Respiratory Infection in Mice.

Bacteria can be motile and planktonic or, alternatively, sessile and participating in the biofilm mode of growth. The transition between these lifestyles can be regulated by a second messenger, cyclic dimeric GMP (c-di-GMP). High intracellular c-di-GMP concentration correlates with biofilm formation and motility inhibition in most bacteria, including Bordetella bronchiseptica, which causes respiratory tract infections in mammals and forms biofilms in infected mice. We previously described the diguanylate cyclase BdcA as involved in c-di-GMP synthesis and motility regulation in B. bronchiseptica; here, we further describe the mechanism whereby BdcA is able to regulate motility and biofilm formation. Amino acid replacement of GGDEF with GGAAF in BdcA is consistent with the conclusion that diguanylate cyclase activity is necessary for biofilm formation and motility regulation, although we were unable to confirm the stability of the mutant protein. In the absence of the bdcA gene, B. bronchiseptica showed enhanced motility, strengthening the hypothesis that BdcA regulates motility in B. bronchiseptica We showed that c-di-GMP-mediated motility inhibition involved regulation of flagellin expression, as high c-di-GMP levels achieved by expressing BdcA significantly reduced the level of flagellin protein. We also demonstrated that protein BB2109 is necessary for BdcA activity, motility inhibition, and biofilm formation. Finally, absence of the bdcA gene affected bacterial infection, implicating BdcA-regulated functions as important for bacterium-host interactions. This work supports the role of c-di-GMP in biofilm formation and motility regulation in B. bronchiseptica, as well as its impact on pathogenesis.IMPORTANCE Pathogenesis of Bordetella spp., like that of a number of other pathogens, involves biofilm formation. Biofilms increase tolerance to biotic and abiotic factors and are proposed as reservoirs of microbes for transmission to other organs (trachea, lungs) or other hosts. Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a second messenger that regulates transition between biofilm and planktonic lifestyles. In Bordetella bronchiseptica, high c-di-GMP levels inhibit motility and favor biofilm formation. In the present work, we characterized a B. bronchiseptica diguanylate cyclase, BdcA, which regulates motility and biofilm formation and affects the ability of B. bronchiseptica to colonize the murine respiratory tract. These results provide us with a better understanding of how B. bronchiseptica can infect a host.