Streptococcus pyogenes Polymyxin B-Resistant Mutants Display Enhanced ExPortal Integrity

Abstract

The ExPortal protein secretion organelle in Streptococcus pyogenes is an anionic phospholipid-containing membrane microdomain enriched in Sec translocons and postsecretion protein biogenesis factors. Polymyxin B binds to and disrupts ExPortal integrity, resulting in defective secretion of several toxins. To gain insight into factors that influence ExPortal organization, a genetic screen was conducted to select for spontaneous polymyxin B-resistant mutants displaying enhanced ExPortal integrity. Whole-genome resequencing of 25 resistant mutants revealed from one to four mutations per mutant genome clustered primarily within a core set of 10 gene groups. Construction of mutants with individual deletions or insertions demonstrated that 7 core genes confer resistance and enhanced ExPortal integrity through loss of function, while 3 were likely due to gain of function and/or combinatorial effects. Core resistance genes include a transcriptional regulator of lipid biosynthesis, several genes involved in nutrient acquisition, and a variety of genes involved in stress responses. Two members of the latter class also function as novel regulators of the secreted SpeB cysteine protease. Analysis of the most frequently isolated mutation, a single nucleotide deletion in a track of 9 consecutive adenine residues in pstS, encoding a component of a high-affinity P i transporter, suggests that this sequence functions as a molecular switch to facilitate stress adaptation. Together, these data suggest the existence of a membrane stress response that promotes enhanced ExPortal integrity and resistance to cationic antimicrobial peptides.

Genomic Landscape of Intrahost Variation in Group A Streptococcus: Repeated and Abundant Mutational Inactivation of the fabT Gene Encoding a Regulator of Fatty Acid Synthesis

“…Our result is consistent with recent data showing that mutations in fabT render S. pyogenes more resistant to polymyxin B (55). Lack of FabT could also confer on GAS increased tolerance for acidic conditions, such as those found in necrotic tissue.…”

A PTS EII mutant library in Group A Streptococcus identifies a promiscuous man‐family PTS transporter influencing SLS‐mediated hemolysis

“…In Streptococcus pneumoniae, a Δ manLMN mutant was recently shown to be unable to grow on non-preferred carbohydrates due to the lack of induction of other metabolic operons (Fleming & Camilli, 2016). In contrast, ManLMN has been shown to have a large number of substrates in other bacteria and plays a more direct role in metabolism of these sugars (Port et al , 2014, Tong et al , 2011). The exact mechanism by which the mannose-specific EII affects the metabolism of other carbohydrates in GAS is not known.…”

Streptococcus pyogenes Malate Degradation Pathway Links pH Regulation and Virulence

“…During growth, the plate was shaken every 10 min for 30 s, followed by a 5-s wait period and measurement of the OD 600 . Data were normalized relative to uninoculated medium, and growth rates were calculated as described previously (34). Growth rates are reported as doubling time (t 1/2 ) and were determined from a series of 7 time points collected over a 60-min period that defines the peak rate of growth, which typically occurred prior to the culture reaching 15 to 30% of the max OD 600 .…”

“…5A and B). To verify that this growth defect was specific for malate utilization and not a general defect under all conditions, strains were also grown in unmodified C medium, as well as in C medium supplemented with 0.2% maltose (a non-PTS sugar) (34). Comparisons of final yield from overnight cultures demonstrate that growth of both the PtsI -and HPr H15A mutants is similar to the WT in unmodified medium (Fig.…”