Biology subjects

Bose, J. L.

Publications and source records attributed to Bose, J. L..

4 recordsLinked to original sources

bioRxiv2024

The Staphylococcus aureus small non-coding RNA IsrR regulates TCA cycle activity and virulence

Staphylococcus aureus has evolved mechanisms to cope with low iron (Fe) availability in host tissues. S. aureus uses the ferric uptake transcriptional regulator (Fur) to sense titers of cytosolic Fe. Upon Fe depletion, apo-Fur relieves transcriptional repression of genes utilized for Fe uptake. We demonstrate that an S. aureus {Delta}fur mutant has decreased expression of acnA, which codes for the Fe-dependent enzyme aconitase. Decreased acnA expression prevented the {Delta}fur mutant from growing with amino acids as sole carbon and energy sources. Suppressor analysis determined that a mutation in isrR, which produces a regulatory RNA, permitted growth by decreasing isrR transcription. The decreased AcnA activity of the {Delta}fur mutant was partially relieved by an {Delta}isrR mutation. Directed mutation of bases predicted to facilitate the interaction between the acnA transcript and IsrR, decreased the ability of IsrR to control acnA expression in vivo and IsrR bound to the acnA transcript in vitro. IsrR also bound to the transcripts coding the alternate TCA cycle proteins sdhC, mqo, citZ, and citM. Whole cell metal analyses suggest that IsrR promotes Fe uptake and increases intracellular Fe not ligated by macromolecules. Lastly, we determined that Fur and IsrR promote infection using murine skin and acute pneumonia models. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/601953v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1ec27b2org.highwire.dtl.DTLVardef@1224b64org.highwire.dtl.DTLVardef@83cd49org.highwire.dtl.DTLVardef@11a9cea_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Molecular basis for the activation of the Fatty Acid Kinase complex of Staphylococcus aureus

Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. The complex, consisting of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, is known to impact virulence and disease outcomes. However, FAKs structure and enzymatic mechanism remain poorly understood. Here, we used a combination of modeling, biochemical, and cell-based approaches to establish critical details of FAK activity. Solved structures of the apo and ligand-bound FakA kinase domain captured the protein state through ATP hydrolysis. Additionally, targeted mutagenesis of an understudied FakA Middle domain identified critical residues within a metal-binding pocket that contribute to FakA dimer stability and protein function. Regarding the complex, we demonstrated nanomolar affinity between FakA and FakB and generated computational models of the complexs quaternary structure. Together, these data provide critical insight into the structure and function of the FAK complex which is essential for understanding its mechanism.

microbiology↗

bioRxiv2023

Fructose-1-kinase has pleiotropic roles in Escherichia coli

In Escherichia coli, the master transcription regulator Catabolite Repressor Activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a {Delta}fruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. The {Delta}fruK strain also alters biofilm formation. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other {gamma}-proteobacteria, which conserve both Cra and FruK.

microbiology↗

bioRxiv2020

Controlling the Growth of the Skin Commensal Staphylococcus epidermidis Using D-Alanine Auxotrophy

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics, or genetic elements conferring antibiotic resistance, enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied D-alanine. The S. epidermidis strain, NRRL B-4268 {Delta}alr1{Delta}alr2{Delta}dat (SE{Delta}{Delta}{Delta}) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the D-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in D-alanine deficient media, pooled human blood, and skin. In the presence of D-alanine, SE{Delta}{Delta}{Delta} colonized and increased expression of human {beta}-defensin 2 in cultured human skin models in vitro. SE{Delta}{Delta}{Delta}, showed a low propensity to revert to D-alanine prototrophy, and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SE{Delta}{Delta}{Delta} as a live biotherapeutic strain whose growth can be controlled by D-alanine.

microbiology↗