Pilus Adhesin RrgA Interacts with Complement Receptor 3, Thereby Affecting Macrophage Function and Systemic Pneumococcal Disease

10.1128/mBio.00535-12Pneumococcal pili have been shown to influence pneumococcal colonization, disease development, and the inflammatory response in mice. The role of the pilus-associated RrgA adhesin in pneumococcal interactions with murine and human macrophages was investigated. Expression of pili with RrgA enhanced the uptake of pneumococci by murine and human macrophages that was abolished by antibodies to complement receptor 3 (CR3) and not seen in CR3-deficient macrophages. Recombinant RrgA, but not pilus subunit RrgC, promoted CR3-mediated phagocytosis of coated beads by murine and human macrophages. Flow cytometry showed that purified CR3 binds pneumococcal cells expressing RrgA, and purified RrgA was shown to interact with CR3 and its I domain. In vivo, RrgA facilitated spread of pneumococci from the upper airways and peritoneal cavity to the bloodstream. Earlier onset of septicemia and more rapidly progressing disease was observed in wild-type mice compared to CR3-deficient mice challenged intranasally or intraperitoneally with pneumococci. Motility assays and time-lapse video microscopy showed that pneumococcal stimulation of macrophage motility required RrgA and CR3. These findings, together with the observed RrgA-dependent increase of intracellular survivors up to 10 h following macrophage infection, suggest that RrgA-CR3-mediated phagocytosis promotes systemic pneumococcal spread from local sites.IMPORTANCE Streptococcus pneumoniae is a major contributor to morbidity and mortality in infectious diseases globally. Symptomatology is mainly due to pneumococcal interactions with host cells leading to an inflammatory response. However, we still need more knowledge on how pneumococci talk to immune cells and the importance of this interaction. Recently, a novel structure was identified on the pneumococcal surface, an adhesive pilus found in about 30% of clinical pneumococcal isolates. The pilus has been suggested to be important for successful spread of antibiotic-resistant pneumococcal clones globally. Here we sought to identify mechanisms for how the pneumococcal pilin subunit RrgA contributes to disease development by interacting with host immune cells. Our data suggest a new way for how pneumococci may cross talk with phagocytic cells and affect disease progression. An increased understanding of these processes may lead to better strategies for how to treat these common infections.Peer reviewe

Thioredoxin 1 Promotes Intracellular Replication and Virulence of Salmonella enterica Serovar Typhimurium

The effect of the cytoplasmic reductase and protein chaperone thioredoxin 1 on the virulence of Salmonella enterica serovar Typhimurium was evaluated by deleting the trxA, trxB, or trxC gene of the cellular thioredoxin system, the grxA or gshA gene of the glutathione/glutaredoxin system, or the dsbC gene coding for a thioredoxin-dependent periplasmic disulfide bond isomerase. Mutants were tested for tolerance to oxidative and nitric oxide donor substances in vitro, for invasion and intracellular replication in cultured epithelial and macrophage-like cells, and for virulence in BALB/c mice. In these experiments only the gshA mutant, which was defective in glutathione synthesis, exhibited sensitization to oxidative stress in vitro and a small decrease in virulence. In contrast, the trxA mutant did not exhibit any growth defects or decreased tolerance to oxidative or nitric oxide stress in vitro, yet there were pronounced decreases in intracellular replication and mouse virulence. Complementation analyses using defined catalytic variants of thioredoxin 1 showed that there is a direct correlation between the redox potential of thioredoxin 1 and restoration of intracellular replication of the trxA mutant. Attenuation of mouse virulence that was caused by a deficiency in thioredoxin 1 was restored by expression of wild-type thioredoxin 1 in trans but not by expression of a catalytically inactive variant. These results clearly imply that in S. enterica serovar Typhimurium, the redox-active protein thioredoxin 1 promotes virulence, whereas in vitro tolerance to oxidative stress depends on production of glutathione

Thioredoxin 1 Participates in the Activity of the Salmonella enterica Serovar Typhimurium Pathogenicity Island 2 Type III Secretion System ▿

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium relies on its Salmonella pathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in a trxA mutant. Complementation analyses using wild-type trxA or a genetically engineered trxA coding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections

Salicylidene Acylhydrazides That Affect Type III Protein Secretion in Salmonella enterica Serovar Typhimurium▿

A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector β-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1. Salicylidene acylhydrazides also affected intracellular bacterial replication in murine macrophage-like cells and blocked the transport of an epitope-tagged SPI2 effector protein. Two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We conclude that salicylidene acylhydrazides affect bacterial T3SS activity in S. enterica and hence could be used as lead substances when designing specific inhibitors of bacterial T3SSs in order to pharmaceutically intervene with bacterial virulence

FasL and TRAIL Induce Epidermal Apoptosis and Skin Ulceration Upon Exposure to Leishmania major

Receptor-mediated apoptosis is proposed as an important regulator of keratinocyte homeostasis in human epidermis. We have previously reported that Fas/FasL interactions in epidermis are altered during cutaneous leishmaniasis (CL) and that keratinocyte death through apoptosis may play a pathogenic role for skin ulceration. To further investigate the alterations of apoptosis during CL, a keratinocyte cell line (HaCaT) and primary human epidermal keratinocytes were incubated with supernatants from Leishmania major-infected peripheral blood mononuclear cells. An apoptosis-specific microarray was used to assess mRNA expression in HaCaT cells exposed to supernatants derived from L. major-infected cultures. Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression were significantly up-regulated, and apoptosis was detected in both HaCaT and human epidermal keratinocyte cells. The keratinocyte apoptosis was partly inhibited through blocking of Fas or FasL and even more efficiently through TRAIL neutralization. Up-regulation of Fas on keratinocytes in epidermis and the presence of FasL-expressing macrophages and T cells in dermis were previously reported by us. In this study, keratinocytes expressing TRAIL, as well as the proapoptotic receptor TRAIL-R2, were detected in skin biopsies from CL cases. We propose that activation of Fas and TRAIL apoptosis pathways, in the presence of inflammatory mediators at the site of infection, leads to tissue destruction and ulceration during CL