The heme-responsive PrrH sRNA regulates Pseudomonas aeruginosa pyochelin gene expression.

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron for growth and virulence, yet this nutrient is sequestered by the innate immune system during infection. When iron is limiting, P. aeruginosa expresses the PrrF1 and PrrF2 small RNAs (sRNAs), which post-transcriptionally repress expression of nonessential iron-containing proteins, thus sparing this nutrient for more critical processes. The genes for the PrrF1 and PrrF2 sRNAs are arranged in tandem on the chromosome, allowing for the transcription of a longer heme-responsive sRNA, termed PrrH. While the functions of PrrF1 and PrrF2 have been extensively studied, the role of PrrH in P. aeruginosa physiology and virulence is not well understood. In this study, we performed transcriptomic and proteomic studies to identify the PrrH regulon. In shaking cultures, the pyochelin synthesis proteins were increased in two distinct prrH mutants compared to the wild type, while the mRNAs for these proteins were not affected by the prrH mutation. We identified complementarity between the PrrH sRNA and the sequence upstream of the pchE mRNA, suggesting the potential for PrrH to directly regulate the expression of genes for pyochelin synthesis. We further showed that pchE mRNA levels were increased in the prrH mutants when grown in static but not shaking conditions. Moreover, we discovered that controlling for the presence of light was critical for examining the impact of PrrH on pchE expression. As such, our study reports on the first likely target of the PrrH sRNA and highlights key environmental variables that will allow for future characterization of PrrH function. IMPORTANCE In the human host, iron is predominantly in the form of heme, which Pseudomonas aeruginosa can acquire as an iron source during infection. We previously showed that the iron-responsive PrrF small RNAs (sRNAs) are critical for mediating iron homeostasis during P. aeruginosa infection; however, the function of the heme-responsive PrrH sRNA remains unclear. In this study, we identified genes for pyochelin siderophore biosynthesis, which mediates uptake of inorganic iron, as a novel target of PrrH regulation. This study therefore highlights a novel relationship between heme availability and siderophore biosynthesis in P. aeruginosa.

Increasing the Capture Rate of Circulating Tumor DNA in Unaltered Plasma Using Passive Microfluidic Mixer Flow Cells.

A limiting factor in using blood-based liquid biopsies for cancer detection is the volume of extracted blood required to capture a measurable number of circulating tumor DNA (ctDNA). To overcome this limitation, we developed a technology named the dCas9 capture system to capture ctDNA from unaltered flowing plasma, removing the need to extract the plasma from the body. This technology has provided the first opportunity to investigate whether microfluidic flow cell design can affect the capture of ctDNA in unaltered plasma. With inspiration from microfluidic mixer flow cells designed to capture circulating tumor cells and exosomes, we constructed four microfluidic mixer flow cells. Next, we investigated the effects of these flow cell designs and the flow rate on the rate of captured spiked-in BRAF T1799A (BRAFMut) ctDNA in unaltered flowing plasma using surface-immobilized dCas9. Once the optimal mass transfer rate of ctDNA, identified by the optimal ctDNA capture rate, was determined, we investigated whether the design of the microfluidic device, flow rate, flow time, and the number of spiked-in mutant DNA copies affected the rate of capture by the dCas9 capture system. We found that size modifications to the flow channel had no effect on the flow rate required to achieve the optimal capture rate of ctDNA. However, decreasing the size of the capture chamber decreased the flow rate required to achieve the optimal capture rate. Finally, we showed that, at the optimal capture rate, different microfluidic designs using different flow rates could capture DNA copies at a similar rate over time. In this study, the optimal capture rate of ctDNA in unaltered plasma was identified by adjusting the flow rate in each of the passive microfluidic mixer flow cells. However, further validation and optimization of the dCas9 capture system are required before it is ready to be used clinically.

The heme-responsive PrrH sRNA regulates Pseudomonas aeruginosa pyochelin gene expression.

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron for growth and virulence, yet this nutrient is sequestered by the innate immune system during infection. When iron is limiting, P. aeruginosa expresses the PrrF1 and PrrF2 small regulatory RNAs (sRNAs), which post-transcriptionally repress expression of non-essential iron-containing proteins thus sparing this nutrient for more critical processes. The genes for the PrrF1 and PrrF2 sRNAs are arranged in tandem on the chromosome, allowing for the transcription of a longer heme-responsive sRNA, termed PrrH. While the functions of PrrF1 and PrrF2 have been studied extensively, the role of PrrH in P. aeruginosa physiology and virulence is not well understood. In this study, we performed transcriptomic and proteomic studies to identify the PrrH regulon. In shaking cultures, the pyochelin synthesis proteins were increased in two distinct prrH mutants compared to wild type, while the mRNAs for these proteins were not affected by prrH mutation. We identified complementarity between the PrrH sRNA and sequence upstream of the pchE mRNA, suggesting potential for PrrH to directly regulate expression of genes for pyochelin synthesis. We further showed that pchE mRNA levels were increased in the prrH mutants when grown in static but not shaking conditions. Moreover, we discovered controlling for the presence of light was critical for examining the impact of PrrH on pchE expression. As such, our study reports on the first likely target of the PrrH sRNA and highlights key environmental variables that will allow for future characterization of PrrH function.

Complete Genome Sequence of Staphylococcus epidermidis CSF41498.

Staphylococcus epidermidis CSF41498 is amenable to genetic manipulation and has been used to study mechanisms of biofilm formation. We report here the whole-genome sequence of this strain, which contains 2,427 protein-coding genes and 82 RNAs within its 2,481,008-bp-long genome, as well as three plasmids.

Transcriptional Regulation of icaADBC by both IcaR and TcaR in Staphylococcus epidermidis.

S. epidermidis is a primary cause of biofilm-mediated infections in humans due to adherence to foreign bodies. A major staphylococcal biofilm accumulation molecule is polysaccharide intracellular adhesin (PIA), which is synthesized by enzymes encoded by the icaADBC operon. Expression of PIA is highly variable among clinical isolates, suggesting that PIA expression levels are selected in certain niches of the host. However, the mechanisms that govern enhanced icaADBC transcription and PIA synthesis in these isolates are not known. We hypothesized that enhanced PIA synthesis in these isolates was due to function of IcaR and/or TcaR. Thus, two S. epidermidis isolates (1457 and CSF41498) with different icaADBC transcription and PIA expression levels were studied. Constitutive expression of both icaR and tcaR demonstrated that both repressors are functional and can completely repress icaADBC transcription in both 1457 and CSF41498. However, it was found that IcaR was the primary repressor for CSF41498 and TcaR was the primary repressor for 1457. Further analysis demonstrated that icaR transcription was repressed in 1457 in comparison to CSF41498, suggesting that TcaR functions as a repressor only in the absence of IcaR. Indeed, DNase I footprinting suggests IcaR and TcaR may bind to the same site within the icaR-icaA intergenic region. Lastly, we found mutants expressing variable amounts of PIA could rapidly be selected from both 1457 and CSF41498. Collectively, we propose that strains producing enhanced PIA synthesis are selected within certain niches of the host through several genetic mechanisms that function to repress icaR transcription, thus increasing PIA synthesis.IMPORTANCEStaphylococcus epidermidis is a commensal bacterium that resides on our skin. As a commensal, it protects humans from bacterial pathogens through a variety of mechanisms. However, it is also a significant cause of biofilm infections due to its ability to bind to plastic. Polysaccharide intercellular adhesin is a significant component of biofilm, and we propose that the expression of this polysaccharide is beneficial in certain host niches, such as providing extra strength when the bacterium is colonizing the lumen of a catheter, and detrimental in others, such as colonization of the skin surface. We show here that fine-tuning of icaADBC transcription, and thus PIA synthesis, is mediated via two transcriptional repressors, IcaR and TcaR.

The metalloprotease SepA governs processing of accumulation-associated protein and shapes intercellular adhesive surface properties in Staphylococcus epidermidis.

The otherwise harmless skin inhabitant Staphylococcus epidermidis is a major cause of healthcare-associated medical device infections. The species' selective pathogenic potential depends on its production of surface adherent biofilms. The Cell wall-anchored protein Aap promotes biofilm formation in S. epidermidis, independently from the polysaccharide intercellular adhesin PIA. Aap requires proteolytic cleavage to act as an intercellular adhesin. Whether and which staphylococcal proteases account for Aap processing is yet unknown. Here, evidence is provided that in PIA-negative S. epidermidis 1457Δica, the metalloprotease SepA is required for Aap-dependent S. epidermidis biofilm formation in static and dynamic biofilm models. qRT-PCR and protease activity assays demonstrated that under standard growth conditions, sepA is repressed by the global regulator SarA. Inactivation of sarA increased SepA production, and in turn augmented biofilm formation. Genetic and biochemical analyses demonstrated that SepA-related induction of biofilm accumulation resulted from enhanced Aap processing. Studies using recombinant proteins demonstrated that SepA is able to cleave the A domain of Aap at residue 335 and between the A and B domains at residue 601. This study identifies the mechanism behind Aap-mediated biofilm maturation, and also demonstrates a novel role for a secreted staphylococcal protease as a requirement for the development of a biofilm.

Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress.

Staphylococcus epidermidis is a leading cause of hospital-associated infections, including those of intravascular catheters, cerebrospinal fluid shunts, and orthopedic implants. Multiple biofilm matrix molecules with heterogeneous characteristics have been identified, including proteinaceous, polysaccharide, and nucleic acid factors. Two of the best-studied components in S. epidermidis include accumulation-associated protein (Aap) and polysaccharide intercellular adhesin (PIA), produced by the enzymatic products of the icaADBC operon. Biofilm composition varies by strain as well as environmental conditions, and strains producing PIA-mediated biofilms are more robust. Clinically, biofilm-mediated infections occur in a variety of anatomical sites with diverse physiological properties. To test the hypothesis that matrix composition exhibits niche specificity, biofilm-related genetic and physical properties were compared between S. epidermidis strains isolated from high-shear and low-shear environments. Among a collection of 105 clinical strains, significantly more isolates from high-shear environments carried the icaADBC operon than did those from low-shear settings (43.9% versus 22.9%, P < 0.05), while there was no significant difference in the presence of aap (77.2% versus 75.0%, P > 0.05). Additionally, a significantly greater number of high-shear isolates were capable of forming biofilm in vitro in a microtiter assay (82.5% versus 45.8%, P < 0.0001). However, even among high-shear clinical isolates, less than half contained the icaADBC locus; therefore, we selected for ica-negative variants with increased attachment to abiotic surfaces to examine PIA-independent biofilm mechanisms. Sequencing of selected variants identified substitutions capable of enhancing biofilm formation in multiple genes, further highlighting the heterogeneity of S. epidermidis biofilm molecules and mechanisms. IMPORTANCEStaphylococcus epidermidis is a leading cause of infections related to biomaterials, mostly due to their ability to form biofilm. Biofilm accumulation mechanisms vary, including those that are dependent on specific proteins, environmental DNA (eDNA), or polysaccharide intercellular adhesin (PIA). We found that those isolates obtained from high-shear environments, such as the lumen of a catheter, are more likely to produce PIA-mediated biofilms than those isolates obtained from a low-shear biomaterial-related infection. This suggests that PIA functions as a mechanism that is protective against shear flow. Finally, we performed selection experiments documenting the heterogeneity of biofilm accumulation molecules that function in the absence of PIA, further documenting the biofilm-forming potential of S. epidermidis.