An abundant periplasmic protein of the denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans is PstS,a component of an ABC phosphate transport system

To understand a physiological role of an abundant 34-kDa periplasmic protein in the denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans grown in a medium containing malate as the carbon source, the gene for the protein was isolated. The deduced amino acid sequence of the protein had a sequence similarity of 66.2% to that of PstS from Sinorhizobium meliloti. The downstream sequence of the Rhodobacter pstS contained five genes similar to pstCAB and phoUB, and its upstream sequence contained a putative regulatory sequence that is analogous to the Pho box involved in phosphate-limitation-induced gene expression in Escherichia coli. Both the amount of the PstS and the pstS promoter-driven expression of lacZ activity increased about two-fold in response to phosphate limitation. This is the first isolation of pst genes encoding proteins of an ABC phosphate transporter system from phototrophic bacteria.     

A proteomic analysis of penicillin resistance in Streptococcus pneumoniae reveals a novel role for PstS, a subunit of the phosphate ABC transporter

Resistance to penicillin is widespread in the Gram-positive bacterium Streptococcus pneumoniae, and while several mutations are known to be implicated in resistance other mechanisms are likely to occur. We used a proteomic screen of two independent mutants in which resistance was selected in vitro. We found a number of differentially expressed proteins including PstS, a subunit of the phosphate ABC transporter of S. pneumoniae. This protein was increased in both mutants, a phenotype correlated to increased RNA expression of the entire phosphate ABC transporter operon. Inactivation of the pstS gene led to increased susceptibility to penicillin in the wild-type strain. To further link the expression of the ABC phosphate transporter with penicillin resistance, we looked at pstS mRNA levels in 12 independent clinical isolates sensitive and resistant to penicillin and found an excellent correlation between resistance and increased expression of pstS. Inactivation of pstS in one of the clinical isolates significantly reduced penicillin resistance. Global approaches are ideally suited for the discovery of novel factors in the biology of resistance.     

An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton.

In the marine cyanobacterium Synechococcus sp. strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P1) availability (D. J. Scanlan, N. H. Mann, and N. G. Carr, Mol. Microbiol. 10:181-191, 1993). We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton. Expression of PstS in Synechococcus sp. strain WH7803 was observed when the P1 concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P1 typical of oligotrophic conditions. PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion. In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples. We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton. Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp. strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding.     

Modulation of lung epithelial functions by Pseudomonas aeruginosa

Microorganisms gain access to the airways and respiratory epithelial surface during normal breathing. Most inhaled microbes are trapped on the mucous layer coating the nasal epithelium and upper respiratory tract, and are cleared by ciliary motion. Microorganisms reaching the alveolar spaces are deposited on the pulmonary epithelium. This contact initiates complex offensive and defensive strategies by both parties. Here, we briefly outline how the pulmonary pathogen Pseudomonas aeruginosa uses multi-pronged strategies that include cell surface appendages, and secreted and injected virulence determinants to switch from an unobtrusive soil bacterium to a pathogen for lung epithelium colonization. Understanding the complex interactions between the lung epithelium and P. aeruginosa might enable more effective therapeutic strategies against infection in cystic fibrosis and immuno-compromised individuals.     

Duplication of genes encoding the immunodominant 38 kDa antigen in Mycobacterium intracellulare

Abstract Mycobacterium avium is a causative agent of mycobacterioses in systemically immunocompromised individuals, whereas Mycobacterium intracellulare is responsible for causing infections in relatively immunocompetent hosts. In an attempt to identify components that could be involved in virulence, we characterised the 38 kDa-encoding gene of M. intracellulare that is absent in M. avium . This antigen cross-reacts immunologically with a major 38 kDa antigen of M. tuberculosis , and both antigens are homologues of the phosphate transport subunit S (PstS) of the pst complex of Escherichia coli . Unlike the M. tuberculosis complex the M. intracellulare coding gene was found to be duplicated. We also identified and characterised other pst genes that may constitute an operon. Considering that multiple isoforms of PstS are present in mycobacteria the possible role of pstS1 genes for pathogenesis is discussed.     

The Phosphate-Binding Protein of Escherichia coli Is Not Essential for Pi-Regulated Expression of the pho Regulon

Disruption of pstS encoding the P(i)-binding protein in Escherichia coli generally leads to the constitutive expression of the pho regulon. We demonstrate that P(i)-controlled expression is restored when the activity of the P(i) transporter PitA or PitB is increased. Apparently, PstS is not an essential component of the signal transduction pathway.     

Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.     

The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression.

The cystic fibrosis gene product, CFTR, and the multidrug resistance P-glycoprotein (encoded by the MDR1 gene) are structurally related proteins and both are associated with epithelial chloride channel activities. We have compared their cell-specific expression in the rat by in situ hybridization. In all tissues examined the two genes were found to have complementary patterns of expression, demonstrating exquisite regulation in both cell-specific and temporal fashions. Additionally, a switch in expression from one gene to the other was observed in certain tissues. For example, expression in the intestine switches from CFTR to MDR1 as the cells migrate across the crypt-villus boundary. A switch from CFTR to MDR1 expression was also observed in the uterine epithelium upon pregnancy. These data suggest that CFTR and P-glycoprotein serve analogous roles in epithelial cells and provide additional evidence that P-glycoprotein has a physiological role in regulating epithelial cell volume. The patterns of expression suggest that the regulation of these two genes is coordinately controlled.     

Expression and polarization of intercellular adhesion molecule-1 on human intestinal epithelia: consequences for CD11b/CD18-mediated interactions with neutrophils.

BACKGROUND: Epithelial dysfunction and patient symptoms in inflammatory intestinal diseases such as ulcerative colitis and Crohn's disease correlate with migration of neutrophils (PMN) across the intestinal epithelium. In vitro modeling of PMN transepithelial migration has revealed distinct differences from transendothelial migration. By using polarized monolayers of human intestinal epithelia (T84), PMN transepithelial migration has been shown to be dependent on the leukocyte integrin CD11b/CD18 (Mac-1), but not on CD11a/CD18 (LFA-1). Since intercellular adhesion molecule-I (ICAM-1) is an important endothelial counterreceptor for these integrins, its expression in intestinal epithelia and role in PMN-intestinal epithelial interactions was investigated. MATERIALS AND METHODS: A panel of antibodies against different domains of ICAM-1, polarized monolayers of human intestinal epithelia (T84), and natural human colonic epithelia were used to examine the polarity of epithelial ICAM-1 surface expression and the functional role of ICAM-1 in neutrophil-intestinal epithelial adhesive interactions. RESULTS: While no surface expression of ICAM-1 was detected on unstimulated T84 cells, interferon-gamma (IFN gamma) elicited a marked expression of ICAM-1 that selectively polarized to the apical epithelial membrane. Similarly, apically restricted surface expression of ICAM-1 was detected in natural human colonic epithelium only in association with active inflammation. With or without IFN gamma pre-exposure, physiologically directed (basolateral-to-apical) transepithelial migration of PMN was unaffected by blocking monoclonal antibodies (mAbs) to ICAM-1. In contrast, PMN migration across IFN gamma-stimulated monolayers in the reverse (apical-to-basolateral) direction was inhibited by anti-ICAM-1 antibodies. Adhesion studies revealed that T84 cells adhered selectively to purified CD11b/CD18 and such adherence, with or without IFN gamma pre-exposure, was unaffected by ICAM-1 mAb. Similarly, freshly isolated epithelial cells from inflamed human intestine bound to CD11b/CD18 in an ICAM-1-independent fashion. CONCLUSIONS: These data indicate that ICAM-1 is strictly polarized in intestinal epithelia and does not represent a counterreceptor for neutrophil CD11b/CD18 during physiologically directed transmigration, but may facilitate apical membrane-PMN interactions after the arrival of PMN in the intestinal lumen.     

Studies on adhesion, haemagglutination and other biological properties of Vibrio cholerae O139

Abstract The adhesive capabilities of eight Vibrio cholerae O139 epidemic strains to isolated rabbit intestinal epithelial cells (RIEC) were observed to be high similar to those observed with a Vibrio cholerae O1 strain isolated from patients. Toxin production by the strains, measured by accumulation of fluid in rabbit ileal loop model, was high and the toxin was lethal as the animal expired within 6 h. Culture filtrates of the strains exhibited the presence of vascular permeability factor which produce induration and necrosis in the adult rabbit and guinea pig skin. All the strains showed high to moderate haemagglutinin titres against chicken erythrocytes and produced El Tor-like haemolysin. SDS-PAGE of the outer membrane preparation of the strains showed the presence of major protein component at 38 kDa region. The lethality of the toxin, high adhesive activity, shifting of the major outer membrane protein band and production of thermolabile haemolysin on Wagatsuma agar were the major variations of these epidemic strains from V. cholerae O1 and V. cholerae non-O1 strains isolated previously.     

Enterocyte death and intestinal barrier maintenance in homeostasis and disease

The intestinal epithelium is the largest surface area of the body in contact with the external environment. This specialized single cell layer is constantly renewed and is a physical barrier that separates intestinal microbiota from underlying tissues, preventing bacterial infiltration and subsequent inflammation. Specialized secretory epithelial cell types such as Paneth cells and goblet cells limit bacterial adhesion and infiltration by secreting antibacterial peptides and mucins, respectively. Rapid cell renewal coincides with apical exfoliation of 'old' enterocytes without compromising epithelial barrier integrity. When the intestinal epithelium is inflamed barrier integrity can be compromised, due to uncontrolled death of enterocytes allowing bacterial infiltration. This review discusses the different mechanisms which regulate or affect intestinal barrier integrity under homeostatic and inflammatory conditions.     

Exploring the rearrangement of sensory intelligence in proteobacteria: insight of <Emphasis Type="Italic">Pho regulon</Emphasis>

Pho regulon is a highly evolved and conserved mechanism across the microbes to fulfil their phosphate need. In this study, 52 proteobacteria genomes were analyzed for the presence of phosphorus acquisition genes, their pattern of arrangement and copy numbers. The diverse genetic architecture of the Pho regulon genes indicates the evolutionary challenge of nutrient limitation, particularly phosphorus, faced by bacteria in their environment. The incongruence between the Pho regulon proteins phylogeny and species phylogeny along with the presence of additional copies of pstS and pstB genes, having cross similarity with other genera, suggest the possibility of horizontal gene transfer event. The substitution rate analysis and multiple sequence alignment of the Pho regulon proteins were analyzed to gain additional insight into the evolution of the Pho regulon system. This comprehensive study confirms that genes perform the regulatory function (phoBR) were vertically inherited, whereas interestingly, genes whose product involved in direct interaction with the environment (pstS) acquired by horizontal gene transfer. The substantial amino acid substitutions in PstS most likely contribute to the successful adaptation of bacteria in different ecological condition dealing with different phosphorus availability. The findings decipher the intelligence of the bacteria which enable them to carry out the targeted alteration of genes to cope up with the environmental condition.     

Regulation of matrilysin expression in airway epithelial cells by Pseudomonas aeruginosa flagellin

Matrilysin (matrix metalloproteinase-7) is expressed by mucosal epithelia throughout the body and functions in host defense by activating murine intestinal alpha-defensins. In normal adult human lung, matrilysin is expressed at low levels in the airway epithelium, but is markedly up-regulated in cystic fibrosis (CF). Because CF lungs support a heavy bacterial load, we assessed if relevant CF pathogens regulate matrilysin expression in human lung epithelial cells. Indeed, acute infection with Pseudomonas aeruginosa (but not Staphylococcus aureus, Haemophilus influenzae, or Klebsiella pneumoniae) induced the expression of matrilysin in Calu-3 lung epithelial cells. Increased matrilysin mRNA levels were detectable at 3 h post-infection and peaked at a 25-fold induction between 6 and 8 h. Both P. aeruginosa CF isolates and laboratory strains induced matrilysin expression to similar levels. Flagellin, the monomeric precursor of bacterial flagella, was identified as the inductive factor released by P. aeruginosa that regulated matrilysin expression. In addition, flagellin-null mutants failed to stimulate matrilysin expression in cultured cells or in lungs infected in vivo. These data show that P. aeruginosa (and specifically flagellin) potently stimulates matrilysin expression in lung epithelial cells and may mediate the overexpression of this proteinase in CF lungs.     

The protective potency of probiotic bacteria and their microbial products against enteric infections-review

The intestinal environment accommodates a wide range of contents ranging from harmless beneficial dietary and microbial flora to harmful pathogenic bacteria. This has resulted in the development of highly adapted epithelial cells lining the intestine. This adaptation involves the potential of crypt cells to proliferate and to constantly replace villous cells that are lost due to maturity or death. As a result, the normal intestinal epithelial integrity and functions are maintained. This phenomenon is eminent in intestinal defense whereby the intestinal epithelial cells serve as a physical barrier against luminal agents. The protection against agents in the gut lumen can only be effective if the epithelium is intact. Restitution of the damaged epithelium is therefore crucial in this type of defense.     

Overexpression of MDR1 in an intestinal cell line results in increased cholesterol uptake from micelles

The multiple drug resistance protein, MDR1, is highly expressed on the apical surface of intestinal epithelial cells. The physiologic substrate of this protein remains unclear. Several studies using compounds known to act as MDR1 inhibitors have suggested that MDR1 may be involved in the transport of cholesterol from the plasma membrane to the endoplasmic reticulum where it is esterified. To examine the role of MDR1 in cholesterol uptake by intestinal cells, the rat intestinal epithelial cell line IEC-18, was stably transfected with human MDR1. MDR1-transfected cells exhibited increased expression of MDR1 protein, reduced accumulation of vinblastine and increased uptake of [(3)H]cholesterol from cholesterol/monolein/taurocholate micelles. These studies provide the first direct evidence that the level of MDR1 expression in intestinal cells can influence the amount of cholesterol taken up by those cells. This is also the first demonstration that a multiple drug resistance protein can function in the net uptake, rather than efflux, of a substrate.     

Gene cloning and expression of the Pseudomonas aeruginosa periplasmic phosphate-binding protein

Abstract The pstS gene, encoding the Pseudomonas aeruginosa phosphate-binding protein, was cloned onto a cosmid vector into Escherichia coli , and localized by subcloning, mapping the insertion site of Tn 501 in a P. aeruginosa pstS ::Tn 501 mutant, and hybridization to an oligonucleotide pool synthesized according to the aminoterminal amino acid sequence of the purified protein. The cloned pstS gene was transferred to P. aeruginosa pstS mutants. The P. aeruoginosa phosphate-binding protein was also expressed and secreted into the periplasm of E. coli pstS mutants.     

Modulation of stem cell fate in intestinal homeostasis,injury and repair

The mammalian intestinal epithelium constitutes the largest barrier against the external environment and makes flexible responses to various types of stimuli. Epithelial cells are fast-renewed to counteract constant damage and disrupted barrier function to maintain their integrity. The homeostatic repair and regeneration of the intestinal epithelium are governed by the Lgr5⁺ intestinal stem cells (ISCs) located at the base of crypts, which fuel rapid renewal and give rise to the different epithelial cell types. Protracted biological and physicochemical stress may challenge epithelial integrity and the function of ISCs. The field of ISCs is thus of interest for complete mucosal healing, given its relevance to diseases of intestinal injury and inflammation such as inflammatory bowel diseases. Here, we review the current understanding of the signals and mechanisms that control homeostasis and regeneration of the intestinal epithelium. We focus on recent insights into the intrinsic and extrinsic elements involved in the process of intestinal homeostasis, injury, and repair, which fine-tune the balance between self-renewal and cell fate specification in ISCs. Deciphering the regulatory machinery that modulates stem cell fate would aid in the development of novel therapeutics that facilitate mucosal healing and restore epithelial barrier function.     

Intestinal Tissues Induce an SNP Mutation in Pseudomonas aeruginosa That Enhances Its Virulence: Possible Role in Anastomotic Leak

The most feared complication following intestinal resection is anastomotic leakage. In high risk areas (esophagus/rectum) where neoadjuvant chemoradiation is used, the incidence of anastomotic leaks remains unacceptably high (～10%) even when performed by specialist surgeons in high volume centers. The aims of this study were to test the hypothesis that anastomotic leakage develops when pathogens colonizing anastomotic sites become in vivo transformed to express a tissue destroying phenotype. We developed a novel model of anastomotic leak in which rats were exposed to pre-operative radiation as in cancer surgery, underwent distal colon resection and then were intestinally inoculated with Pseudomonas aeruginosa, a common colonizer of the radiated intestine. Results demonstrated that intestinal tissues exposed to preoperative radiation developed a significant incidence of anastomotic leak (>60%; p<0.01) when colonized by P. aeruginosa compared to radiated tissues alone (0%). Phenotype analysis comparing the original inoculating strain (MPAO1- termed P1) and the strain retrieved from leaking anastomotic tissues (termed P2) demonstrated that P2 was altered in pyocyanin production and displayed enhanced collagenase activity, high swarming motility, and a destructive phenotype against cultured intestinal epithelial cells (i.e. apoptosis, barrier function, cytolysis). Comparative genotype analysis between P1 and P2 revealed a single nucleotide polymorphism (SNP) mutation in the mexT gene that led to a stop codon resulting in a non-functional truncated protein. Replacement of the mutated mexT gene in P2 with mexT from the original parental strain P1 led to reversion of P2 to the P1 phenotype. No spontaneous transformation was detected during 20 passages in TSB media. Use of a novel virulence suppressing compound PEG/Pi prevented P. aeruginosa transformation to the tissue destructive phenotype and prevented anastomotic leak in rats. This work demonstrates that in vivo transformation of microbial pathogens to a tissue destroying phenotype may have important implications in the pathogenesis of anastomotic leak.     

The DING family of proteins: ubiquitous in eukaryotes,but where are the genes?

PstS and DING proteins are members of a superfamily of secreted, high‐affinity phosphate‐binding proteins. Whereas microbial PstS have a well‐defined role in phosphate ABC transporters, the physiological function of DING proteins, named after their DINGGG N termini, still needs to be determined. PstS and DING proteins co‐exist in some Pseudomonas strains, to which they confer a highly adhesive and virulent phenotype. More than 30 DING proteins have now been purified, mostly from eukaryotes. They are often associated with infections or with dysregulation of cell proliferation. Consequently, eukaryotic DING proteins could also be involved in cell–cell communication or adherence. The ubiquitous presence in eukaryotes of proteins structurally and functionally related to bacterial virulence factors is intriguing, as is the absence of eukaryotic genes encoding DING proteins in databases. DING proteins in eukaryotes could originate from unidentified commensal or symbiotic bacteria and could contribute to essential functions. Alternatively, DING proteins could be encoded by eukaryotic genes sharing special features that prevent their cloning. Both hypotheses are discussed.