PspA-mediated aggregation protects Streptococcus pneumoniae against desiccation on fomites.

IMPORTANCE: Spn is a dangerous human pathogen capable of causing pneumonia and invasive disease. The virulence factor PspA has been studied for nearly four decades with well-established roles in pneumococcal evasion of C-reactive protein and neutralization of lactoferricin. Herein, we show that mammalian (m)GAPDH in mucosal secretions promotes aggregation of pneumococci in a PspA-dependent fashion, whereas lactoferrin counters this effect. PspA-mediated GAPDH-dependent bacterial aggregation protected Spn in nasal lavage elutes and grown in vitro from desiccation on fomites. Furthermore, surviving pneumococci within these aggregates retained their ability to colonize naïve hosts after desiccation. We report that Spn binds to and forms protein complexes on its surface composed of PspA, mGAPDH, and lactoferrin. Changes in the levels of these proteins therefore most likely have critical implications on Spn colonization, survival on fomites, and transmission.

PspA-mediated aggregation protects Streptococcus pneumoniae against desiccation on fomites.

Streptococcus pneumoniae (Spn) resides in the nasopharynx where it can disseminate to cause disease. One key Spn virulence factor is pneumococcal surface protein A (PspA), which promotes survival by blocking the antimicrobial peptide lactoferricin. PspA has also been shown to mediate attachment to dying epithelial cells in the lower airway due to its binding of cell surface-bound mammalian (m)GAPDH. Importantly, the role of PspA during colonization is not well understood. Wildtype Spn was present in nasal lavage elutes collected from asymptomatically colonized mice at levels ~10-fold higher that its isogenic PspA-deficient mutant (ΔpspA). Wildtype Spn also formed aggregates in mucosal secretions composed of sloughed epithelial cells and hundreds of pneumococci, whereas ΔpspA did not. Spn within the center of these aggregates better survived prolonged desiccation on fomites than individual pneumococci and were capable of infecting naïve mice, indicating PspA-mediated aggregation conferred a survival/transmission advantage. Incubation of Spn in saline containing mGAPDH also enhanced tolerance to desiccation, but only for wildtype Spn. mGAPDH was sufficient to cause low-level aggregation of wildtype Spn but not ΔpspA. In strain WU2, the subdomain of PspA responsible for binding GAPDH (aa230-281) is ensconced within the lactoferrin (LF)-binding domain (aa167-288). We observed that LF inhibited GAPDH-mediated aggregation and desiccation tolerance. Using surface plasmon resonance, we determined that Spn forms multimeric complexes of PspA-GAPDH-LF on its surface and that LF dislodges GAPDH. Our findings have important implications regarding pneumococcal colonization/transmission processes and ongoing PspA-focused immunization efforts for this deadly pathogen.

A Jack of All Trades: The Role of Pneumococcal Surface Protein A in the Pathogenesis of Streptococcus pneumoniae.

Streptococcus pneumoniae (Spn), or the pneumococcus, is a Gram-positive bacterium that colonizes the upper airway. Spn is an opportunistic pathogen capable of life-threatening disease should it become established in the lungs, gain access to the bloodstream, or disseminate to vital organs including the central nervous system. Spn is encapsulated, allowing it to avoid phagocytosis, and current preventative measures against infection include polyvalent vaccines composed of capsular polysaccharide corresponding to its most prevalent serotypes. The pneumococcus also has a plethora of surface components that allow the bacteria to adhere to host cells, facilitate the evasion of the immune system, and obtain vital nutrients; one family of these are the choline-binding proteins (CBPs). Pneumococcal surface protein A (PspA) is one of the most abundant CBPs and confers protection against the host by inhibiting recognition by C-reactive protein and neutralizing the antimicrobial peptide lactoferricin. Recently our group has identified two new roles for PspA: binding to dying host cells via host-cell bound glyceraldehyde 3-phosphate dehydrogenase and co-opting of host lactate dehydrogenase to enhance lactate availability. These properties have been shown to influence Spn localization and enhance virulence in the lower airway, respectively. Herein, we review the impact of CBPs, and in particular PspA, on pneumococcal pathogenesis. We discuss the potential and limitations of using PspA as a conserved vaccine antigen in a conjugate vaccine formulation. PspA is a vital component of the pneumococcal virulence arsenal - therefore, understanding the molecular aspects of this protein is essential in understanding pneumococcal pathogenesis and utilizing PspA as a target for treating or preventing pneumococcal pneumonia.

High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex function.

The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the ß-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF's partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494L and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.

Improper Coordination of BamA and BamD Results in Bam Complex Jamming by a Lipoprotein Substrate.

The ß-barrel assembly machinery, the Bam complex, is central to the biogenesis of integral outer membrane proteins (OMPs) as well as OMP-dependent surface-exposed lipoproteins, such as regulator of capsule synthesis protein F (RcsF). Previous genetic analysis established the model that nonessential components BamE and BamB have overlapping, redundant functions to enhance the kinetics of the highly conserved BamA/BamD core. Here we report that BamE plays a specialized nonredundant role in the Bam complex required for surface exposure of RcsF. We show that the lack of bamE, but not bamB, completely abolishes assembly of RcsF/OMP complexes and establish that the inability to assemble RcsF/OMP complexes is a molecular reason underlying all synthetic lethal interactions of ΔbamE Our genetic analysis and biochemical cross-linking suggest that RcsF accumulates on BamA when BamA cannot engage with BamD because of its limited availability or the incompatible conformation. The role of BamE is to promote proper coordination of RcsF-bound BamA with BamD to complete OMP assembly around RcsF. We show that in the absence of BamE, RcsF is stalled on BamA, thus blocking its function, and we identify the lipoprotein RcsF as a bona fide jamming substrate of the Bam complex.IMPORTANCE The ß-barrel assembly machinery, the Bam complex, consists of five components, BamA to -E, among which BamA and BamD are highly conserved and essential. The nonessential components are believed to play redundant roles simply by improving the rate of ß-barrel folding. Here we show that BamE contributes a specific and nonoverlapping function to the Bam complex. BamE coordinates BamA and BamD to form a complex between the lipoprotein RcsF and its partner outer membrane ß-barrel protein, allowing RcsF to reach the cell surface. In the absence of BamE, RcsF accumulates on BamA, thus blocking the activity of the Bam complex. As the Bam complex is a major antibiotic target in Gram-negative bacteria, the discovery that a lipoprotein can act as a jamming substrate may open the door for development of novel Bam complex inhibitors.

Negative Control of RpoS Synthesis by the sRNA ReaL in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen, able to resist host defense mechanisms and antibiotic treatment. In Pae, the master regulator of stress responses RpoS (σS) is involved in the regulation of quorum sensing and several virulence genes. Here, we report that the sRNA ReaL translationally silences rpoS mRNA, which results in a decrease of the RpoS levels. Our studies indicated that ReaL base-pairs with the Shine-Dalgarno region of rpoS mRNA. These studies are underlined by a highly similar transcription profile of a rpoS deletion mutant and a reaL over-expressing strain.

The Anaerobically Induced sRNA PaiI Affects Denitrification in Pseudomonas aeruginosa PA14.

Pseudomonas aeruginosa is an opportunistic pathogen that can thrive by anaerobic respiration in the lungs of cystic fibrosis patients using nitrate as terminal electron acceptor. Here, we report the identification and characterization of the small RNA PaiI in the P. aeruginosa strain 14 (PA14). PaiI is anaerobically induced in the presence of nitrate and depends on the two-component system NarXL. Our studies revealed that PaiI is required for efficient denitrification affecting the conversion of nitrite to nitric oxide. In the absence of PaiI anaerobic growth was impaired on glucose, which can be reconciled with a decreased uptake of the carbon source under these conditions. The importance of PaiI for anaerobic growth is further underlined by the observation that a paiI deletion mutant was impaired in growth in murine tumors.

Cross-regulation by CrcZ RNA controls anoxic biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA) can thrive in anaerobic biofilms in the lungs of cystic fibrosis (CF) patients. Here, we show that CrcZ is the most abundant PA14 RNA bound to the global regulator Hfq in anoxic biofilms grown in cystic fibrosis sputum medium. Hfq was crucial for anoxic biofilm formation. This observation complied with an RNAseq based transcriptome analysis and follow up studies that implicated Hfq in regulation of a central step preceding denitrification. CrcZ is known to act as a decoy that sequesters Hfq during relief of carbon catabolite repression, which in turn alleviates Hfq-mediated translational repression of catabolic genes. We therefore inferred that CrcZ indirectly impacts on biofilm formation by competing for Hfq. This hypothesis was supported by the findings that over-production of CrcZ mirrored the biofilm phenotype of the hfq deletion mutant, and that deletion of the crcZ gene augmented biofilm formation. To our knowledge, this is the first example where competition for Hfq by CrcZ cross-regulates an Hfq-dependent physiological process unrelated to carbon metabolism.

RNASeq Based Transcriptional Profiling of Pseudomonas aeruginosa PA14 after Short- and Long-Term Anoxic Cultivation in Synthetic Cystic Fibrosis Sputum Medium.

The opportunistic human pathogen Pseudomonas aeruginosa can thrive under microaerophilic to anaerobic conditions in the lungs of cystic fibrosis patients. RNASeq based comparative RNA profiling of the clinical isolate PA14 cultured in synthetic cystic fibrosis medium was performed after planktonic growth (OD600 = 2.0; P), 30 min after shift to anaerobiosis (A-30) and after anaerobic biofilm growth for 96h (B-96) with the aim to reveal differentially regulated functions impacting on sustained anoxic biofilm formation as well as on tolerance towards different antibiotics. Most notably, functions involved in sulfur metabolism were found to be up-regulated in B-96 cells when compared to A-30 cells. Based on the transcriptome studies a set of transposon mutants were screened, which revealed novel functions involved in anoxic biofilm growth.In addition, these studies revealed a decreased and an increased abundance of the oprD and the mexCD-oprJ operon transcripts, respectively, in B-96 cells, which may explain their increased tolerance towards meropenem and to antibiotics that are expelled by the MexCD-OprD efflux pump. The OprI protein has been implicated as a target for cationic antimicrobial peptides, such as SMAP-29. The transcriptome and subsequent Northern-blot analyses showed that the abundance of the oprI transcript encoding the OprI protein is strongly decreased in B-96 cells. However, follow up studies revealed that the susceptibility of a constructed PA14ΔoprI mutant towards SMAP-29 was indistinguishable from the parental wild-type strain, which questions OprI as a target for this antimicrobial peptide in strain PA14.