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Staphylococcus aureus is an opportunistic pathogen that can cause life-threatening infections, particularly in immunocompromised individuals. The high-level virulence of S. aureus largely relies on its diverse and variable collection of virulence factors and immune evasion proteins, including the six serine protease-like proteins SplA to SplF. Spl proteins are expressed by most clinical isolates of S. aureus, but little is known about the molecular mechanisms by which these proteins modify the host's immune response for the benefit of the bacteria. Here, we identify SplB as a protease that inactivates central human complement proteins, i.e., C3, C4, and the activation fragments C3b and C4b, by preferentially cleaving their α-chains. SplB maintained its proteolytic activity in human serum, degrading C3 and C4. SplB further cleaved the components of the terminal complement pathway, C5, C6, C7, C8, and C9. In contrast, the important soluble human complement regulators factor H and C4b-binding protein (C4BP), as well as C1q, were left intact. Thereby, SplB reduced C3b-mediated opsonophagocytosis by human neutrophils as well as C5b-9 deposition on the bacterial surface. In conclusion, we identified the first physiological substrates of the S. aureus extracellular protease SplB. This enzyme inhibits all three complement pathways and blocks opsonophagocytosis. Thus, SplB can be considered a novel staphylococcal complement evasion protein. IMPORTANCE The success of bacterial pathogens in immunocompetent humans depends on the control and inactivation of host immunity. S. aureus, like many other pathogens, efficiently blocks host complement attack early in infection. Aiming to understand the role of the S. aureus-encoded orphan proteases of the Spl operon, we asked whether these proteins play a role in immune escape. We found that SplB inhibits all three complement activation pathways as well as the lytic terminal complement pathway. This blocks the opsonophagocytosis of the bacteria by neutrophils. We also clarified the molecular mechanisms: SplB cleaves the human complement proteins C3, C4, C5, C6, C7, C8, and C9 as well as factor B but not the complement inhibitors factor H and C4BP. Thus, we identify the first physiological substrates of the extracellular protease SplB of S. aureus and characterize SplB as a novel staphylococcal complement evasion protein.

Escherichia coli and many other bacterial species can enter into a viable but nonculturable (VBNC) state, which is a survival strategy adopted by cells exposed to adverse environmental conditions. Pyruvate is known to be one factor that promotes resuscitation of VBNC cells. Here we studied the role of a pyruvate-sensing network, composed of the histidine kinase-response regulator systems BtsS/BtsR and YpdA/YpdB and the target gene btsT , encoding the high-affinity pyruvate/H + symporter BtsT, in the resuscitation of VBNC E. coli K-12 cells after exposure to cold for 120 days. Analysis of the proteome of VBNC cells revealed upregulation, relative to exponentially growing cells, of BtsT and other proteins involved in pyruvate metabolism. Provision of pyruvate stimulated protein and DNA biosynthesis, and thus resuscitation, in wild-type but not btsSR ypdAB mutant VBNC cells. This result was corroborated by time-dependent tracking of the resuscitation of individual VBNC E. coli cells observed in a microfluidic system. Finally, transport assays revealed that 14 C-labeled pyruvate was rapidly taken up into VBNC cells by BtsT. These results provide the first evidence that pyruvate is taken up as a carbon source for the resuscitation of VBNC E. coli cells. IMPORTANCE Viable but nonculturable (VBNC) bacteria do not form colonies in standard medium but otherwise retain their metabolic activity and can express toxic proteins. Many bacterial genera, including Escherichia , Vibrio , and Listeria , have been shown to enter the VBNC state upon exposure to adverse conditions, such as low temperature, radiation, and starvation. Ultimately, these organisms pose a public health risk with potential implications for the pharmaceutical and food industries, as dormant organisms are especially difficult to selectively eliminate and VBNC bacteria can be resuscitated if placed in an environment with appropriate nutrition and temperature. Here we used a microfluidic system to monitor the resuscitation of single VBNC cells over time. We provide new molecular insights into the initiation of resuscitation by demonstrating that VBNC E. coli cells rapidly take up pyruvate with an inducible high-affinity transporter, whose expression is triggered by the BtsSR-YpdAB sensing network., (Copyright © 2019 American Society for Microbiology.)

The peptide transporter carbon starvation (CstA) family (transporter classification [TC] 2.A.114) belongs to the second largest superfamily of secondary transporters, the amino acid/polyamine/organocation (APC) superfamily. No representative of the CstA family has previously been characterized either biochemically or structurally, but we have now identified the function of one of its members, the transport protein YjiY of Escherichia coli Expression of the yjiY gene is regulated by the LytS-like histidine kinase BtsS, a sensor of extracellular pyruvate, together with the LytTR-like response regulator BtsR. YjiY consists of 716 amino acids, which form 18 putative transmembrane helices. Transport studies with intact cells provided evidence that YjiY is a specific and high-affinity transporter for pyruvate ( K m , 16 μM). Furthermore, reconstitution of the purified YjiY into proteoliposomes revealed that YjiY is a pyruvate/H + symporter. It has long been assumed that E. coli possesses a transporter(s) for pyruvate, but the present study is the first to definitively identify such a protein. Based on its function, we propose to change the name of the uncharacterized gene yjiY to btsT for Brenztraubensäure (the German word for pyruvate) transporter. IMPORTANCE BtsT (formerly known as YjiY) is found in many commensal and pathogenic representatives of the Enterobacteriaceae This study for the first time characterizes a pyruvate transporter in E. coli , BtsT, as a specific pyruvate/H + symporter. When nutrients are limiting, BtsT takes up pyruvate from the medium, thus enabling it to be used as a carbon source for the growth and survival of E. coli ., (Copyright © 2017 American Society for Microbiology.)

Fluctuating environments and individual physiological diversity force bacteria to constantly adapt and optimize the uptake of substrates. We focus here on two very similar two-component systems (TCSs) of Escherichia coli belonging to the LytS/LytTR family: BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB. Both TCSs respond to extracellular pyruvate, albeit with different affinities, typically during postexponential growth, and each system regulates expression of a single transporter gene, yjiY and yhjX , respectively. To obtain insights into the biological significance of these TCSs, we analyzed the activation of the target promoters at the single-cell level. We found unimodal cell-to-cell variability; however, the degree of variance was strongly influenced by the available nutrients and differed between the two TCSs. We hypothesized that activation of either of the TCSs helps individual cells to replenish carbon resources. To test this hypothesis, we compared wild-type cells with the btsSR ypdAB mutant under two metabolically modulated conditions: protein overproduction and persister formation. Although all wild-type cells were able to overproduce green fluorescent protein (GFP), about half of the btsSR ypdAB population was unable to overexpress GFP. Moreover, the percentage of persister cells, which tolerate antibiotic stress, was significantly lower in the wild-type cells than in the btsSR ypdAB population. Hence, we suggest that the BtsS/BtsR and YpdA/YpdB network contributes to a balancing of the physiological state of all cells within a population. IMPORTANCE Histidine kinase/response regulator (HK/RR) systems enable bacteria to respond to environmental and physiological fluctuations. Escherichia coli and other members of the Enterobacteriaceae possess two similar LytS/LytTR-type HK/RRs, BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB, which form a functional network. Both systems are activated in response to external pyruvate, typically when cells face overflow metabolism during post-exponential growth. Single-cell analysis of the activation of their respective target genes yjiY and yhjX revealed cell-to-cell variability, and the range of variation was strongly influenced by externally available nutrients. Based on the phenotypic characterization of a btsSR ypdAB mutant compared to the parental strain, we suggest that this TCS network supports an optimization of the physiological state of the individuals within the population., (Copyright © 2017 American Society for Microbiology.)