Streptococcus pneumoniae (S. pneumoniae) causes high early mortality in pneumococcal pneumonia, which is characterized by acute lung injury (ALI). The molecular mechanisms underlying ALI and the high early mortality remain unknown. Despite recent studies that identify deubiquitinating enzyme cylindromatosis (CYLD) as a key regulator for T cell development, tumor cell proliferation, and NF-kappaB transcription factor signaling, its role in regulating bacteria-induced lethality, however, is unknown. Here, we showed that CYLD deficiency protected mice from S. pneumoniae pneumolysin (PLY)-induced ALI and lethality. CYLD was highly induced by PLY, and it inhibited MKK3-p38 kinase-dependent expression of plasminogen activator inhibitor-1 (PAI-1) in lung, thereby potentiating ALI and mortality. Thus, CYLD is detrimental for host survival, thereby indicating a mechanism underlying the high early mortality of pneumococcal pneumonia.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Using a tissue culture system, invasive strains of P. aeruginosa were discovered to induce apoptosis at high frequency in HeLa and other epithelial and fibroblast cell lines. This apoptotic phenotype in the infected cells was determined by several criteria including (i) visual changes in cell morphology, (ii) induction of chromatin condensation and nuclear marginalization, (iii) the presence of a high percentage of cells with subG1 DNA content, and (iv) activation of caspase-3 activity. Induction of the type III secretion machinery, but not invasion of P. aeruginosa is required for induction of apoptosis. The apoptosis phenotype is independent of the cytoskeletal rearrangements that occur in the host cell early after infection. Mutants in P. aeruginosa exoS fail to induce apoptosis and complementation with wild-type exoS restored the apoptosis-inducing capacity, demonstrating that ExoS is the effector molecule. Analysis of exoS activity mutants shows that the ADP-ribosylating capacity of ExoS is essential for inducing the apoptotic pathway.

DsbA is a periplasmic thiol:disulfide oxidoreductase which contributes to the process of protein folding by catalyzing the formation of disulfide bonds. In this study, we demonstrate that the dsbA gene is required for the expression of the type III secretion system under low-calcium inducing conditions, intracellular survival of P. aeruginosa upon infection of HeLa cells, and twitching motility. The diverse phenotypes of the dsbA mutant are likely due to its defect in the folding of proteins that are involved in various biological processes, such as signal sensing, protein secretion, and defense against host clearing. In light of its effect on various virulence factors, DsbA could be an important target for the control of P. aeruginosa infections.

Epithelial cells represent the first line of host innate defense against invading microbes by elaborating a range of molecules involved in pathogen clearance. In particular, epithelial mucins facilitate the mucociliary clearance by physically trapping inhaled microbes. Up-regulation of mucin production thus represents an important host innate defense response against invading microbes. How mucin is induced in upper respiratory Streptococcus pneumoniae infections is unknown. In this study, we show that pneumolysin is required for up-regulation of MUC5AC mucin via TLR4-dependent activation of ERK in human epithelial cells in vitro and in mice in vivo. Interestingly, a “second wave” of ERK activation appears to be important in mediating MUC5AC induction. Moreover, IκB kinase (IKK) α and IKKβ are distinctly involved in MUC5AC induction via an ERK1-dependent, but IκBα-p65- and p100-p52-independent, mechanism, thereby revealing novel roles for IKKs in mediating up-regulation of MUC5AC mucin by S. pneumoniae.

Non-typeable Haemophilus influenza (NTHi) is an important human pathogen causing respiratory tract infections in both adults and children. NTHi infections are characterized by inflammation, which is mainly mediated by nuclear transcription factor kappaB (NF-κB)-dependent production of inflammatory mediators. The deubiquitinating enzyme cylindromatosis (CYLD), loss of which was originally reported to cause a benign human syndrome called cylindromatosis, has been identified as a key negative regulator for NF-κB in vitro. However, little is known about the role of CYLD in bacteria-induced inflammation in vivo. Here, we provided direct evidence for the negative role of CYLD in NTHi-induced inflammation of the mice in vivo. Our data demonstrated that CYLD is induced by NTHi in the middle ear and lung of mice. NTHi-induced CYLD, in turn, negatively regulates NTHi-induced NF-κB activation through deubiquitinating TRAF6 and 7 and down-regulates inflammation. Our data thus indicate that CYLD acts as a negative regulator for NF-κB-dependent inflammation in vivo, hence protecting the host against detrimental inflammatory response to NTHi infection.

Transforming growth factor-b (TGF-b) family members are multifunctional growth factors involved in regulating diverse biological processes. Despite the critical role for TGF-b in regulating cell proliferation, differentiation, migration and development, its role in regulating NF-jBdependent inflammatory response still remains unclear. Here, we show that TGF-b1 induces acetylation of NF-jB p65 subunit to synergistically enhance bacterium nontypeable Haemophilus influenzae-induced NF-jB activation and inflammatory response in vitro and in vivo. The TGF-b1-induced acetylation of p65 is mediated via a Smad3/4-PKA-p300-dependent signaling pathway. Acetylation of p65 at lysine 221 by TGF-b1 is critical for synergistic enhancement of bacteria-induced DNA-binding activity, NF-jB activation, NF-jB-dependent transcription of TNF-a and IL-1b and interstitial polymorphonuclear neutrophil infiltration in vitro and in vivo. These studies provide new insights into the novel regulation of NF-jB by TGF-b signaling.

SummaryWe have previously reported on the isolation of in vivo inducible genes of Pseudomonas aeruginosa using IVET system. One of such genes isolated from burn mouse infection model encodes a short open reading frame with unknown function. In this study, we demonstrate that this gene product specifically suppresses the expression of type III secretion genes in P. aeruginosa , thus named PtrA (Pseudomonas type III repressor A). A direct interaction between the PtrA and type III transcriptional activator ExsA was demonstrated, suggesting that its repressor function is probably realized through inhibition of the ExsA protein function. Indeed, an elevated expression of the exsA compensates the repressor effect of the PtrA. Interestingly, expression of the ptrA is highly and specifically induced by copper cation. A copperresponsive two-component regulatory system, copRcopS , has also been identified and shown to be essential for the copper resistance in P. aeruginosa as well as the activation of ptrA in response to the copper signal. Elevated expression of the ptrA during the infection of mouse burn wound suggests that P. aeruginosa has evolved tight regulatory systems to shut down energy-expensive type III secretion apparatus in response to specific environmental signals, such as copper stress.

The type III secretion system of Pseudomonas aeruginosa is tightly regulated by various environmental signals, such as low calcium and contact with the host cell. However, the exact signals triggering type III secretion are unknown. The present study describes the finding that secretion of P. aeruginosa type III effector molecules requires protein factors from serum and L broth, designated type III secretion factors (TSFs), in addition to the low-calcium environment. In the absence of TSF or calcium chelator EGTA, basal levels of type III effector molecules are accumulated intracellularly. Addition of TSF and EGTA together effectively triggers the secretion of pre-existing effector molecules in a short time, even before the active expression of type III genes; thus, active type III gene expression does not seem to be a prerequisite for type III secretion. A search for TSF molecules in serum and L broth resulted in the identification of albumin and casein as the functional TSF molecules. Although there is no clear sequence similarity between albumin and casein, both proteins are known to have a low-affinity, high-capacity calcium-binding property. Tests of well-studied calcium-binding proteins seemed to indicate that low-affinity calcium-binding proteins have TSF activity, although the requirement of low-affinity calcium-binding ability for the TSF activity is not clear. P. aeruginosa seems to have evolved a sensing mechanism to detect target cells for type III injection through host-derived proteins in combination with a low-calcium signal. Disruption of the bacterial ability to sense low calcium or TSF might be a valid avenue to the effective control of this bacterial pathogen. INTRODUCTIONThe remarkable ability of Pseudomonas aeruginosa to adapt to and thrive in a wide variety of environments contributes significantly to the ability of this bacterium to cause various human infections (Bodey et al., 1983;Holder, 1993;Pier, 2002). Unlike most human pathogens possessing highly restricted host ranges, P. aeruginosa is pathogenic not only to humans, but also to Caenorhabditis elegans, Drosophila and Arabidopsis thaliana (Rahme et al., 1995;D'Argenio et al., 2001;Aballay & Ausubel, 2002). This broad host range is contributed in part by the numerous virulence factors encoded by P. aeruginosa, including exotoxin A, phospholipase C, alkaline protease, elastase, alginate, pyocyanin, pili and non-pilus adhesins (Ramphal et al., 1991;Deretic et al., 1995;Lory & Strom, 1997;Vasil & Ochsner, 1999). P. aeruginosa, like many other Gram-negative animal and plant pathogens, also encodes a type III secretion machinery, where over 30 proteins assemble into a complex designed to deliver effector molecules directly into the cytoplasmic compartment of eukaryotic cells (Yahr et al., 1995;Frank, 1997). Injection of the bacterial effector molecules into the host cells results in various physiological changes, all of which seem to confer a survival advantage on the bacterial pathogen within the host environment (Hueck, 1998;Muller et al...