The influx of neutrophils to infection sites is a fundamental step in host defenses against the frequent human pathogen group B Streptococcus (GBS) and other extracellular bacteria. Using a mouse model of GBS-induced peritonitis, we show in this study that the chemokines Cxcl1 and Cxcl2 play distinctive roles in enhancing the recruitment and the antibacterial activities of neutrophils in a manner that is linked to differences in the cellular sources of these mediators. Cell depletion experiments demonstrated that neutrophils make a significant contribution to the in vivo production of Cxcl2 but not Cxcl1. In vitro, neutrophils responded weakly to LPS but released high levels of Cxcl2 after stimulation with GBS or other bacteria. Neutrophil-derived Cxcl2 acted in an autocrinous manner to increase its own production and to enhance antibacterial activities, including the release of oxygen radicals. In both neutrophils and macrophages, the production of Cxcl1/2 largely required the presence of functional UNC93B1, a chaperone protein involved in signaling by endosomal TLRs. Moreover, the phenotype of UNC93B1-defective phagocytes could be recapitulated by the simultaneous absence of TLR7, 9, and 13 but not by the absence of individual TLRs. Collectively, our data show that neutrophils recognize Gram-positive and Gram-negative bacteria by means of multiple phagosomal TLRs, resulting in de novo synthesis of Cxcl2, amplification of neutrophil recruitment, and potentiation of their antibacterial activities. These data may be useful to devise alternative therapeutic strategies aimed at enhancing the recruitment and the functional activities of polymorphonuclear leukocytes during infections caused by antibiotic-resistant bacteria.

Streptococcus pneumoniae (or pneumococcus) is a highly prevalent human pathogen. Toll-like receptors (TLRs) function as immune sensors that can trigger host defenses against this bacterium. Defects in TLR-activated signaling pathways, including deficiency in the adaptor protein myeloid differentiation factor 88 (MyD88), are associated with markedly increased susceptibility to infection. However, the individual MyD88-dependent TLRs predominantly involved in antipneumococcal defenses have not been identified yet. Here we find that triple knockout mice simultaneously lacking TLR7, TLR9, and TLR13, which sense the presence of bacterial DNA (TLR9) and RNA (TLR7 and TLR13) in the phagolysosomes of phagocytic cells, display a phenotype that largely resembles that of MyD88-deficient mice and rapidly succumb to pneumococcal pneumonitis due to defective neutrophil influx into the lung. Accordingly, TLR7/9/13 triple knockout resident alveolar macrophages were largely unable to respond to pneumococci with the production of neutrophil-attracting chemokines and cytokines. Mice with single deficiencies of TLR7, TLR9, or TLR13 showed unaltered ability to control lung infection but were moderately more susceptible to encephalitis, in association with a decreased ability of microglia to mount cytokine responses in vitro. Our data point to a dominant, tissue-specific role of nucleic acid-sensing pathways in innate immune recognition of S. pneumoniae and also show that endosomal TLRs are largely capable of compensating for the absence of each other, which seems crucial to prevent pneumococci from escaping immune recognition. These results may be useful to develop novel strategies to treat infections by antibiotic-resistant pneumococci based on stimulation of the innate immune system. IMPORTANCE The pneumococcus is a bacterium that frequently causes infections in the lungs, ears, sinus cavities, and meninges. During these infections, body defenses are triggered by tissue-resident cells that use specialized receptors, such as Toll-like receptors (TLRs), to sense the presence of bacteria. We show here that pneumococci are predominantly detected by TLRs that are located inside intracellular vacuoles, including endosomes, where these receptors can sense the presence of nucleic acids released from ingested bacteria. Mice that simultaneously lacked three of these receptors (specifically, TLR7, TLR9, and TLR13) were extremely susceptible to lung infection and rapidly died after inhalation of pneumococci. Moreover, tissue-resident macrophages from these mice were impaired in their ability to respond to the presence of pneumococci by producing inflammatory mediators capable of recruiting polymorphonuclear leucocytes to infection sites. This information may be useful to develop drugs to treat pneumococcal infections, particularly those caused by antibiotic-resistant strains.

Identification of the receptors involved in innate immune recognition of Staphylococcus aureus, a major cause of morbidity and mortality in humans, is essential to develop alternative strategies to treat infections caused by antibiotic-resistant strains. In the current study, we examine the role of endosomal TLRs, which sense the presence of prokaryotic-type nucleic acids, in anti-staphylococcal host defenses using infection models involving genetically defective mice. Single deficiencies in TLR7, 9, or 13 resulted in mild or no decrease in host defenses. However, the simultaneous absence of TLR7, 9, and 13 resulted in markedly increased susceptibility to cutaneous and systemic S. aureus infection concomitantly with decreased production of proinflammatory chemokines and cytokines, neutrophil recruitment to infection sites, and reduced production of reactive oxygen species. This phenotype was significantly more severe than that of mice lacking TLR2, which senses the presence of staphylococcal lipoproteins. Notably, the combined absence of TLR7, 9, and 13 resulted in complete abrogation of IL-12 p70 and IFN-β responses to staphylococcal stimulation in macrophages. Taken together, our data highlight the presence of a highly integrated endosomal detection system, whereby TLR7, 9, and 13 cooperate in sensing the presence of staphylococcal nucleic acids. We demonstrate that the combined absence of these receptors cannot be compensated for by cell surface-associated TLRs, such as TLR2, or cytosolic receptors. These data may be useful to devise strategies aimed at stimulating innate immune receptors to treat S. aureus infections.

Streptococcus agalactiae (group B streptococcus or GBS) is a commensal bacterium that can frequently behave as a pathogen, particularly in the neonatal period and in the elderly. The gut is a primary site of GBS colonization and a potential port of entry during neonatal infections caused by hypervirulent clonal complex 17 (CC17) strains. Here we studied the interactions between the prototypical CC17 BM110 strain and polarized enterocytes using the Caco-2 cell line. GBS could adhere to and invade these cells through their apical or basolateral surfaces. Basolateral invasion was considerably more efficient than apical invasion and predominated under conditions resulting in weakening of cell-to-cell junctions. Bacterial internalization occurred by a mechanism involving caveolae- and lipid raft-dependent endocytosis and actin re-organization, but not clathrin-dependent endocytosis. In the first steps of Caco-2 invasion, GBS colocalized with the early endocytic marker EEA-1, to later reside in acidic vacuoles. Taken together, these data suggest that CC17 GBS selectively adheres to the lateral surface of enterocytes from which it enters through caveolar lipid rafts using a classical, actin-dependent endocytic pathway. These data may be useful to develop alternative preventive strategies aimed at blocking GBS invasion of the intestinal barrier.

The number of multidrug-resistant bacteria is rapidly spreading worldwide. Among the various mechanisms determining resistance to antimicrobial agents, multidrug efflux pumps play a noteworthy role because they export extraneous and noxious substrates from the inside to the outside environment of the bacterial cell contributing to multidrug resistance (MDR) and, consequently, to the failure of anti-infective therapies. The expression of multidrug efflux pumps can be under the control of transcriptional regulators and two-component systems (TCS). TCS are a major mechanism by which microorganisms sense and reply to external and/or intramembrane stimuli by coordinating the expression of genes involved not only in pathogenic pathways but also in antibiotic resistance. In this review, we describe the influence of TCS on multidrug efflux pump expression and activity in some Gram-negative and Gram-positive bacteria. Taking into account the strict correlation between TCS and multidrug efflux pumps, the development of drugs targeting TCS, alone or together with already discovered efflux pump inhibitors, may represent a beneficial strategy to contribute to the fight against growing antibiotic resistance.

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The binding of Streptococcus pneumoniae to collagen is likely an important step in the pathogenesis of pneumococcal infections, but little is known of the underlying molecular mechanisms. Streptococcal surface repeats (SSURE) are highly conserved protein domains present in cell wall adhesins from different Streptococcus species. We find here that SSURE repeats of the pneumococcal adhesin plasminogen and fibronectin binding protein B (PfbB) bind to various types of collagen. Moreover, deletion of the pfbB gene resulted in a significant impairment of the ability of encapsulated or unencapsulated pneumococci to bind collagen. Notably, a PfbB SSURE domain is also bound to the complement component C1q that bears a collagen‐like domain and promotes adherence of pneumococci to host cells by acting as a bridge between bacteria and epithelial cells. Accordingly, deletion of PfbB or pre‐treatment with anti‐SSURE antibodies markedly decreased pneumococcal binding to C1q as well as C1q‐dependent adherence to epithelial and endothelial cells. Further data indicated that C1q promotes pneumococcal adherence by binding to integrin α2β1. In conclusion, our results indicate that the SSURE domains of the PfbB protein promote interactions of pneumococci with various types of collagen and with C1q. These repeats may be useful targets in strategies to control S. pneumoniae infections.

Two-component signaling systems (TCSs) are finely regulated mechanisms by which bacteria adapt to environmental conditions by modifying the expression of target genes. In bacterial pathogenesis, TCSs play important roles in modulating adhesion to mucosal surfaces, resistance to antibiotics, and metabolic adaptation. In the context of urinary tract infections (UTI), one of the most common types infections causing significant health problems worldwide, uropathogens use TCSs for adaptation, survival, and establishment of pathogenicity. For example, uropathogens can exploit TCSs to survive inside bladder epithelial cells, sense osmolar variations in urine, promote their ascension along the urinary tract or even produce lytic enzymes resulting in exfoliation of the urothelium. Despite the usefulness of studying the function of TCSs in in vitro experimental models, it is of primary necessity to study bacterial gene regulation also in the context of host niches, each displaying its own biological, chemical, and physical features. In light of this, the aim of this review is to provide a concise description of several bacterial TCSs, whose activity has been described in mouse models of UTI.