Biology subjects

Callegan, M. C.

Publications and source records attributed to Callegan, M. C..

3 recordsLinked to original sources

bioRxiv2020

Condensins are essential for Pseudomonas aeruginosa corneal virulence through their control of phenotypic programs

Pseudomonas aeruginosa is a significant opportunistic pathogen responsible for a variety of human infections. Its high pathogenicity resides in a diverse array of virulence factors and an ability to adapt to hostile environments. We report that these factors are tied to the activity of condensins, SMC and MksBEF, which primarily function in structural chromosome maintenance. This study revealed that both proteins are required for P. aeruginosa virulence during corneal infection. The reduction in virulence was traced to broad changes in gene expression. Transcriptional signatures of smc and mksB mutants were largely dissimilar and non-additive, with the double mutant displaying a distinct gene expression profile. Affected regulons included those responsible for lifestyle control, primary metabolism, surface adhesion and biofilm growth, iron and sulfur assimilation, and denitrification. Additionally, numerous virulence factors were affected, including type 3 and type 6 secretion systems, hemagglutinin, pyocin and macroglobulin production, and a host of virulence regulators. in vitro properties of condensin mutants mirrored their transcriptional profiles. MksB-deficient cells were impaired in pyocyanin, c-di-GMP production, and sessile growth whereas smc mutants mildly upregulated c-di-GMP, secreted fewer proteases and were growth deficient under nutrient-limiting conditions. Moreover, condensin mutants displayed an abnormal regulation upon transition to stationary phase. These data reveal that condensins are integrated into the control of multiple genetic programs related to epigenetic and virulent behavior, establishing condensins as an essential factor in P. aeruginosa ocular infections. Author SummaryBacterial pathogenicity is a complex phenomenon dependent on the ability of a bacterium to thrive in a hostile environment while combating the host using an array of virulence factors. This study reports that pathogenicity is also tied to structural chromosome maintenance through condensins, proteins that are responsible for the global organization of the chromosome. We show that the two Pseudomonas aeruginosa condensins, SMC and MksB, act as global regulators of gene expression. The inactivation of SMC and MksB induces opposite regulatory programs in the cell that resemble those observed during the acute and chronic phases of infection. A substantial portion of this regulation is mediated by the intracellular signaling network of P. aeruginosa. Accordingly, virulence regulation is altered in condensin mutants. The results were validated by genetic, phenotypic and virulence studies of condensin mutants. Overall, these data establish condensins as an essential factor during ocular P. aeruginosa infections revealing their involvement in the regulatory virulence network and the control of the bacterial lifestyle.

microbiology↗

bioRxiv2020

The Bacillus Virulome in Endophthalmitis

Bacillus cereus is recognized as a causative agent of gastrointestinal syndromes, but can also cause a devastating form of intraocular infection known as endophthalmitis. We have previously reported that the PlcR/PapR master virulence factor regulator system regulates intraocular virulence, and that the S-layer protein (SlpA) contributes to the severity of B. cereus endophthalmitis. To begin to better understand the role of other B. cereus virulence genes in endophthalmitis, expression levels of a subset of factors was measured at the midpoint of disease progression in a murine model of experimental endophthalmitis by RNA-Seq. Several cytolytic toxins were expressed at significantly higher levels in vivo than in BHI. The virulence regulators codY, gntR, and nprR were also expressed in vivo. However, at this timepoint, plcR/papR was not detectable, we previously reported that a B. cereus mutant deficient in PlcR was attenuated in the eye. The motility-related genes fla, fliF, and motB, and the chemotaxis-related gene cheA were detected during infection. We have shown previously that motility and chemotaxis phenotypes are important in B. cereus endophthalmitis. The sodA2 variant of manganese superoxide dismutase was the most highly expression gene in vivo, suggesting that this gene is criticial for intraocular survival, potentially through inhibition of neutrophil activity. Expression of the surface layer protein gene, slpA, an activator of Toll-like receptors (TLR) -2 and -4, and a potent contributor to intraocular inflammation and disease severvity, was also detected during infection, albeit at low levels. In summary, genes expressed in a mouse model of Bacillus endophthalmitis might prove to play crucial roles in the unique virulence of B. cereus endophthalmitis, and serve as candidates for novel therapies designed attenuate the severity of this often blinding infection. Impact statementB. cereus causes a potent and rapid infection of the eye that usually results in blindness or enucleation, even with the utilization of current treatment modalities. This necessitates the development of new treatment modalities based on new targets. To begin to better define those B. cereus factors with roles in intraocular infection, we analyzed the expression of genes with both known and hypothesized roles in intraocular infection at the midpoint of infection using a murine model of Bacillus endophthalmitis. Potentially targetable candidate genes were demonstrated to be expressed in vivo, which suggests that these genes might contribute to the unique virulence of B. cereus endophthalmitis. Importantly, our results begin to define the virulome of B. cereus in intraocular infections and identify previously uncharacterized factors with potential roles in the severity and outcome of Bacillus endophthalmitis.

microbiology↗

bioRxiv2020

Expression of Bacillus cereus Virulence-Related Genes in an Ocular Infection-Related Environment

Bacillus cereus produces many factors linked to pathogenesis and is recognized for causing gastrointestinal toxemia and infections. B. cereus also causes a fulminant and often blinding intraocular infection called endophthalmitis. We reported that the PlcR/PapR system regulates intraocular virulence, but the specific factors that contribute to B. cereus virulence in the eye remain elusive. Here, we compared gene expression in ex vivo vitreous humor with expression in Luria Bertani (LB) and Brain Heart Infusion (BHI) broth by RNA-Seq. The expression of several cytolytic toxins in vitreous was less than or similar to levels observed in BHI or LB. Regulators of virulence genes, including PlcR/PapR, were expressed in vitreous. PlcR/PapR was expressed at low levels, though we had reported that PlcR-deficient B. cereus was attenuated in the eye. Chemotaxis and motility genes were expressed at similar levels in LB and BHI, but at low to undetectable levels in vitreous, although motility is an important phenotype for B. cereus in the eye. Superoxide dismutase, a potential inhibitor of neutrophil activity in the eye during infection, was the most highly expressed gene in vitreous. Genes previously reported to be important to intraocular virulence were expressed at low levels in vitreous under these conditions, possibly because in vivo cues are required for higher level expression. Genes expressed in vitreous may contribute to the unique virulence of B. cereus endophthalmitis, and future analysis of the B. cereus virulome in the eye will identify those expressed in vivo, which could potentially be targeted to arrest virulence. Impact statementB. cereus is the causative agent of gastrointestinal infections, but can also cause a serious infection of the eye that often results in blindness or enucleation. Current therapeutic measures often fail to mitigate these poor outcomes. This necessitates the development of new treatment modalities based on new targets. To begin to better define those B. cereus factors with roles in intraocular infection, we analyzed the expression of genes related to gastrointestinal infections, as well as those with both known and hypothesized roles in intraocular infections, after growth in an ex vivo vitreous. Potentially targetable candidate genes were demonstrated to be expressed in vitreous, which suggests that these genes might contribute to the unique virulence of B. cereus endophthalmitis. Importantly, our results lay the groundwork for assessing the expression of these genes in vivo and defining the virulome of B. cereus in intraocular infections.

microbiology↗