Global 30-day morbidity and mortality of surgery for perforated peptic ulcer: GRACE study.

BACKGROUND: There is little international data on morbidity and mortality of surgery for perforated peptic ulcer (PPU). This study aimed to understand the global 30-day morbidity and mortality of patients undergoing surgery for PPU and to identify variables associated with these. METHOD: We performed an international study of adults (≥ 18 years) who underwent surgery for PPU from 1st January 2022 to 30th June 2022. Patients who were treated conservatively or had an underlying gastric cancer were excluded. Patients were divided into subgroups according to age (≤ 50 and > 50 years) and time from onset of symptoms to hospital presentation (≤ 24 and > 24 h). Univariate and Multivariate analyses were carried out to identify factors associated with higher 30-day morbidity and mortality. RESULTS:  1874 patients from 159 centres across 52 countries were included. 78.3% (n = 1467) of the patients were males and the median (IQR) age was 49 years (25). Thirty-day morbidity and mortality were 48.5% (n = 910) and 9.3% (n = 174) respectively. Median (IQR) hospital stay was 7 (5) days. Open surgery was performed in 80% (n = 1505) of the cohort. Age > 50 years [(OR = 1.7, 95% CI 1.4-2), (OR = 4.7, 95% CI 3.1-7.6)], female gender [(OR = 1.8, 95% CI 1.4-2.3), (OR = 1.9, 95% CI 1.3-2.9)], shock on admission [(OR = 2.1, 95% CI 1.7-2.7), (OR = 4.8, 95% CI 3.2-7.1)], and acute kidney injury [(OR = 2.5, 95% CI 1.9-3.2), (OR = 3.9), 95% CI 2.7-5.6)] were associated with both 30-day morbidity and mortality. Delayed presentation was associated with 30-day morbidity [OR = 1.3, 95% CI 1.1-1.6], but not mortality. CONCLUSIONS: This study showed that surgery for PPU was associated with high 30-day morbidity and mortality rate. Age, female gender, and signs of shock at presentation were associated with both 30-day morbidity and mortality.

Genomic and functional characterization of a mucosal symbiont involved in early-stage colorectal cancer.

Colorectal cancer is a major health concern worldwide. Growing evidence for the role of the gut microbiota in the initiation of CRC has sparked interest in approaches that target these microorganisms. However, little is known about the composition and role of the microbiota associated with precancerous polyps. Here, we found distinct microbial signatures between patients with and without polyps and between polyp subtypes using sequencing and culturing techniques. We found a correlation between Bacteroides fragilis recovered and the level of inflammatory cytokines in the mucosa adjacent to the polyp. Additional analysis revealed that B. fragilis from patients with polyps are bft-negative, activate NF-κB through Toll-like receptor 4, induce a pro-inflammatory response, and are enriched in genes associated with LPS biosynthesis. This study provides fundamental insight into the microbial microenvironment of the pre-neoplastic polyp by highlighting strain-specific genomic and proteomic differences, as well as more broad compositional differences in the microbiome.

A pandemic recap: lessons we have learned.

On January 2020, the WHO Director General declared that the outbreak constitutes a Public Health Emergency of International Concern. The world has faced a worldwide spread crisis and is still dealing with it. The present paper represents a white paper concerning the tough lessons we have learned from the COVID-19 pandemic. Thus, an international and heterogenous multidisciplinary panel of very differentiated people would like to share global experiences and lessons with all interested and especially those responsible for future healthcare decision making. With the present paper, international and heterogenous multidisciplinary panel of very differentiated people would like to share global experiences and lessons with all interested and especially those responsible for future healthcare decision making.

Clinically Healthy Human Gingival Tissues Show Significant Inter-individual Variability in GCF Chemokine Expression and Subgingival Plaque Microbial Composition.

Aim: Clinically healthy gingival tissue is maintained through controlled regulation of host defense mechanisms against plaque biofilm overgrowth. One key component is the transit of neutrophils from the vasculature into gingival tissue where the expression of different neutrophil chemokines are tightly regulated. This cross-sectional study examines the inter-individual variability in chemokine profiles within gingival crevicular fluid (GCF) in relation to the subgingival bacterial community in a state of gingival health. Methods: Gingival crevicular fluid and subgingival plaque samples were collected from mesiobuccal surfaces of all six Ramfjord teeth of 20 systemically healthy individuals (14.55 ± 1.67 years). A multiplex immunoassay was carried out to quantify the expression of 40 different chemokines in the healthy gingival tissue. Neutrophils were assessed indirectly by myeloperoxidase (MPO) in GCF using traditional ELISA. Characterization of healthy subgingival plaque was conducted with the Illumina Miseq targeting the 16S rRNA gene. Results: In health, there are distinct variations within individual gingival crevicular fluid chemokine expression profiles, as well as in the concentration of neutrophils, that divided the participants into high or low chemokine expressing groups. Specifically, key differences were identified within MIF (2683.54 ± 985.82 pg per 30-s sample), IL-8/CXCL8 (170.98 ± 176.96 pg per 30-s sample), Gro-α/CXCL1 (160.42 ± 94.21 pg per 30-s sample), ENA-78/CXCL5 (137.76 ± 76.02 pg per 30-s sample), IL-1ß (51.39 ± 37.23 pg per 30-s sample), TNF-α (1.76 ± 1.79 pg per 30-s sample), and IFN-γ (0.92 ± 0.54 pg per 30-s sample). Of these identified chemokines, the highest correlation was associated between IL-8/CXCL8 and neutrophils (r = 0.54, p = 0.014). Furthermore, species characterization of healthy subgingival plaque revealed significant inter-individual variability that identified two unique groups unrelated to the previously identified chemokine groups. Conclusion: The lack of concordance between the microbial composition and chemokine profile during health may be a reflection of the unique microbial composition of each individual coupled with variations within their host response, emphasizing the vast complexity of the defense mechanisms in place to maintain gingival health.

The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the fimB Allele and Gingipain Activity.

The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1ß (IL-1ß) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A→T), creating a premature stop codon in the 33277 fimB open reading frame relative to the 381 fimB allele. Gene exchange experiments established that the 33277 fimB allele reduces the immune-stimulatory capacity of this strain. Transcriptome comparisons revealed that multiple genes related to carboxy-terminal domain (CTD) family proteins, including the gingipains, were upregulated in 33277 relative to 381. A gingipain substrate degradation assay demonstrated that cell surface gingipain activity is higher in 33277, and an isogenic mutant strain deficient for the gingipains exhibited an increased ability to induce TLR2 signaling and IL-1ß production. Furthermore, 33277 and 381 mutant strains lacking CTD cell surface proteins were more immune-stimulatory than the parental wild-type strains, consistent with an immune-suppressive role for the gingipains. Our data show that the combination of an intact fimB allele and limited cell surface gingipain activity in P. gingivalis 381 renders this strain more immune-stimulatory. Conversely, a defective fimB allele and high-level cell surface gingipain activity reduce the capacity of P. gingivalis 33277 to stimulate host cell innate immune responses. In summary, genomic and transcriptomic comparisons identified key virulence characteristics that confer divergent host cell innate immune responses to these highly related P. gingivalis strains.

Identification of PGN_1123 as the Gene Encoding Lipid A Deacylase, an Enzyme Required for Toll-Like Receptor 4 Evasion, in Porphyromonas gingivalis.

Removal of one acyl chain from bacterial lipid A by deacylase activity is a mechanism used by many pathogenic bacteria to evade the host's Toll-like receptor 4 (TLR4)-mediated innate immune response. In Porphyromonas gingivalis, a periodontal pathogen, lipid A deacylase activity converts a majority of the initially synthesized penta-acylated lipid A, a TLR4 agonist, to tetra-acylated structures, which effectively evade TLR4 sensing by being either inert or antagonistic at TLR4. In this paper, we report successful identification of the gene that encodes the P. gingivalis lipid A deacylase enzyme. This gene, PGN_1123 in P. gingivalis 33277, is highly conserved within P. gingivalis, and putative orthologs are phylogenetically restricted to the Bacteroidetes phylum. Lipid A of ΔPGN_1123 mutants is penta-acylated and devoid of tetra-acylated structures, and the mutant strain provokes a strong TLR4-mediated proinflammatory response, in contrast to the negligible response elicited by wild-type P. gingivalis Heterologous expression of PGN_1123 in Bacteroides thetaiotaomicron promoted lipid A deacylation, confirming that PGN_1123 encodes the lipid A deacylase enzyme.IMPORTANCE Periodontitis, commonly referred to as gum disease, is a chronic inflammatory condition that affects a large proportion of the population. Porphyromonas gingivalis is a bacterium closely associated with periodontitis, although how and if it is a cause for the disease are not known. It has a formidable capacity to dampen the host's innate immune response, enabling its persistence in diseased sites and triggering microbial dysbiosis in animal models of infection. P. gingivalis is particularly adept at evading the host's TLR4-mediated innate immune response by modifying the structure of lipid A, the TLR4 ligand. In this paper, we report identification of the gene encoding lipid A deacylase, a key enzyme that modifies lipid A to TLR4-evasive structures.

Retrieval of a retained video capsule endoscope with laparoscopic surgery.

Capsule endoscopy (CE) is commonly used for examining and diagnosing gastrointestinal disease, especially small bowel disease. Capsule retention is a well-known and significant complication of CE and requires surgical or endoscopic removal. Most reports described the retrieval of retained CE via laparotomy. We report a case of successful retrieval of the capsule using laparoscopic surgery.

Pharmacobezoar-a rare case presented as gastric outlet obstruction.

BEZOARS are retained concretions of indigestible foreign material that accumulate and conglomerate in the gastrointestinal tract, most commonly in the stomach. Prevalence of bezoar is 0.4%. Bezoars are classified in four categories: phytobezoars; trichobezoars; pharmacobezoars; lactobezoars. A 58-year-old man admitted with complains of pain abdomen and recurrent vomiting since last 3 months. Upper GI endoscopic biopsy reported-chronic gastritis with very occasional non-caseating epitheloid granuloma in lamina propria, no evidence of neoplasia? Crohn's disease. Keeping Crohn's as diagnosis patient was given mesalamine 400 mg tid by gastrophyscian. But patient did not respond so the patient was advised surgical management. Repeat UGI endoscopy revealed multiple pills (mesalamine) in the stomach with gastric outlet obstruction (GOO). Around 40 pills were extracted with the help of flower basket, and then patient develope GOO and underwent Laparoscopic gastrojejunostomy and truncal vagotomy.

Validation of Boey's score in predicting morbidity and mortality in peptic perforation peritonitis in Northwestern India.

BACKGROUND: The major complications of peptic ulcer are hemorrhage, perforation and gastric outlet obstruction with perforation occurring in about 2-10% of patients. Patients with perforated peptic ulcer still have a high rate of morbidity and mortality and to improve the outcomes it is important to stratify the patients into different categories. AIMS: To evaluate the accuracy of Boey scoring system in predicting postoperative morbidity and mortality in patients operated for peptic perforation. METHODS: It was a prospective observational single centre study conducted at SMS Medical College and Hospital, Jaipur, from October 2011 to October 2012 on 180 patients undergoing open surgery for peptic ulcer perforation. Postoperative outcomes in terms of recovery and complications were studied. For prediction of morbidity and mortality by Boey risk stratification, the odds ratio (OR) and 95% confidence interval (95% CI) of each risk score were compared with the outcomes of "0" risk score. RESULTS: The mortality rate increased progressively with increasing numbers of the Boey score: 1.9%, 7.1%, 31.7% and 40% for 0, 1, 2, and 3 scores, respectively (p < 0.001). The morbidity rates for 0, 1, 2, and 3 Boey scores were 13%, 45.7%, 70.7% and 73.3% respectively (p < 0.001). CONCLUSIONS: Boey score is a useful tool for assessing the prognosis of operated cases of peptic perforation and helps in the assessment of mortality and morbidity of these patients.

The Interaction of Implant Luting Cements and Oral Bacteria Linked to Peri-Implant Disease: An In Vitro Analysis of Planktonic and Biofilm Growth--A Preliminary Study.

BACKGROUND: There is little consensus on the most appropriate cement to use when restoring a cement-retained, implant-supported restoration. One consideration should be the interaction of pathogenic oral bacteria with restorative cements. PURPOSE: To determine how oral bacteria associated with peri-implant disease grow in the presence of implant cements. MATERIALS AND METHODS: Five test cements with varying composition (zinc oxide-eugenol [TBO], eugenol-free zinc oxide [TBNE], zinc orthophosphate [FL], and two resin cements [PIC and ML]) were used to fabricate specimen disks. The disks were submerged in bacterial suspensions of either Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, or Porphyromonas gingivalis. Planktonic bacterial growth within the test media was measured by determining the optical density of the cultures (OD600 ). Positive controls (media and bacteria without cement disks) and negative controls (media alone) were similarly evaluated. The mean and standard deviations (SD) were calculated for planktonic growth from three separate experiments. ANOVA statistical analysis with post hoc Tukey tests was performed where differences existed (p < .05). Selected cement disks (TBO and ML) were further examined for bacterial biofilm growth. Surface bacteria were removed and grown on agar media, and colony-forming units (CFUs) were quantified. RESULTS: Planktonic growth for both A. actinomycetemcomitans and P. gingivalis was significantly inhibited (p < .05) when grown in the presence of cement disks consisting of TBNE, PIC, FL, and TBO. In contrast, neither of these bacteria displayed growth inhibition in the presence of ML cement disks. F. nucleatum growth was also significantly inhibited by PIC, FL. and TBO (p < .05), but not by ML and TBNE cement disks. CFU counts for the biofilm study for TBO gave minimal and, in some instances, no bacterial adherence and growth, in contrast to ML, which supported substantially greater bacterial biofilm growth. CONCLUSION: Cements display differing abilities to inhibit both planktonic and biofilm bacterial growth. Cements with the ability to reduce planktonic or biofilm growth of the test bacteria may be advantageous in reducing peri-implant disease. Understanding the microbial growth-inhibiting characteristics of different cement types should be considered important in the selection criteria.

Gastric glomus tumor.

Glomus tumors (GTs) are benign tumors originating from the glomus body which are usually solitary and small lesions. The vast majority are found in the distal extremities, particularly in the hand, wrist, foot and under the fingernails rarely involving visceral organs. Here we report a rare case of gastric GT presented to us with exsanguinating hematemesis and severe anemia. All the initial diagnostic tests were inconclusive. Contrast-enhanced computed tomography abdomen revealed a soft tissue density lesion within the first part of duodenum. Diagnostic laparotomy was planned and a mass of 3 × 2.5 × 2 cm was found at pylorus along greater curvature, without any evidence of lymphadenopathy or metastasis. Distal gastrectomy with gastrojejunostomy was done. Histopathology confirmed the diagnosis of a GT. Immunohistochemistry of tumor cells demonstrated smooth muscle actin and CD34 (very focal).

Adrenocortical carcinoma posing as a pheochromocytoma: a diagnostic dilemma.

Adrenocortical carcinoma (ACC) is a malignant tumour arising from the adrenal cortex, whereas pheochromocytoma is a tumour of the adrenal medulla with occasional presence at extra-adrenal sites. Most of the adrenocortical tumours present clinically with Cushing's syndrome and signs of virilization due to over-production of the respective hormones. It is, however, rare for an adrenocortical tumour to present clinically as a pheochromocytoma. We report the case of a 45-year-old female presenting with clinical symptoms and signs of pheochromocytoma and investigations that resulted in a diagnostic dilemma. The histopathological examination confirmed the presence of ACC after the tumour was excised. This phenomenon was due to the presence of neuroendocrine features of ACC referred to, as a pseudo-pheochromocytoma with extremely limited data in the literature.

A novel class of lipoprotein lipase-sensitive molecules mediates Toll-like receptor 2 activation by Porphyromonas gingivalis.

Infection by the chronic periodontitis-associated pathogen Porphyromonas gingivalis activates a Toll-like receptor 2 (TLR2) response that triggers inflammation in the host but also promotes bacterial persistence. Our aim was to define ligands on the surfaces of intact P. gingivalis cells that determine its ability to activate TLR2. Molecules previously reported as TLR2 agonists include lipopolysaccharide (LPS), fimbriae, the lipoprotein PG1828, and phosphoceramides. We demonstrate that these molecules do not comprise the major factors responsible for stimulating TLR2 by whole bacterial cells. First, P. gingivalis mutants devoid of the reported protein agonists, PG1828 and fimbriae, activate TLR2 as strongly as the wild type. Second, two-phase extraction of whole bacteria resulted in a preponderance of TLR2 agonist activity partitioning to the hydrophilic phase, demonstrating that phosphoceramides are not a major TLR2 ligand. Third, analysis of LPS revealed that TLR2 activation is independent of lipid A structural variants. Instead, activation of TLR2 and TLR2/TLR1 by LPS is in large part due to copurifying molecules that are sensitive to the action of the enzyme lipoprotein lipase. Strikingly, intact P. gingivalis bacterial cells treated with lipoprotein lipase were attenuated in their ability to activate TLR2. We propose that a novel class of molecules comprised by lipoproteins constitutes the major determinants that confer to P. gingivalis the ability to stimulate TLR2 signaling.

In vitro microbiologic evaluation of PTFE and cotton as spacer materials.

OBJECTIVE: To microbiologically evaluate the efficacy of cotton and polytetrafluoroethylene (PTFE) tape used as spacer materials. METHOD AND MATERIALS: Twenty-six extracted human molars were restored using either cotton or PTFE tape as spacers under a standardized provisional restorative material (Cavit). The teeth were incubated for 7 days in a culture of Streptococcus gordonii or in liquid media alone. The spacers were removed and tested for bacterial contamination. The access cavities were also evaluated for bacterial contamination. RESULTS: Nine of 10 teeth with cotton spacers and one of 10 teeth with PTFE spacers were positive for S gordonii growth. The nine teeth in the cotton group also showed contamination of the access cavities. CONCLUSION: Even under optimal conditions, cotton spacers may cause leakage into the access cavities. Cotton fibers may serve as a route for bacterial contamination of the access cavities and root canal space. In contrast, PTFE tape did not provide an avenue for bacterial contamination.

The lipid A phosphate position determines differential host Toll-like receptor 4 responses to phylogenetically related symbiotic and pathogenic bacteria.

The human symbiont Bacteroides thetaiotaomicron promotes intestinal function and health, whereas the phylogenetically related pathogen Porphyromonas gingivalis is associated with the chronic oral inflammatory disease periodontitis. Although both B. thetaiotaomicron and P. gingivalis synthesize lipopolysaccharides (LPS) consisting of penta-acylated, monophosphorylated lipid A in addition to immunologically silent, nonphosphorylated lipid A, they elicit strikingly distinct Toll-like receptor 4 (TLR4) responses. We show that the phosphate position of penta-acylated, monophosphorylated lipid A is a key feature for determining the differential TLR4 responses elicited by these evolutionarily related bacteria. B. thetaiotaomicron produces TLR4-stimulatory lipid A bearing a 1-phosphate, in contrast to P. gingivalis, which produces TLR4-evasive lipid A bearing a 4'-phosphate. Confirming these observations, recombinant Escherichia coli LPS containing penta-acylated, 1-phosphorylated lipid A is more TLR4 stimulatory than LPS containing 4'-phosphorylated lipid A. The specific capacity of a Gram-negative bacterium to alert or evade the host innate immune defense system through TLR4-dependent signaling is currently recognized as a critical aspect defining the relationship between the host and the bacterium. We propose that the distinct lipid A phosphate positions observed for the B. thetaiotaomicron and P. gingivalis LPS contributes to the manifestation of these bacteria as commensal or pathogen within the human host.

AlgK is a TPR-containing protein and the periplasmic component of a novel exopolysaccharide secretin.

The opportunistic pathogen Pseudomonas aeruginosa causes chronic biofilm infections in cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate. Here we show that AlgK, a protein essential for production of high molecular weight alginate, is an outer membrane lipoprotein that contributes to the correct localization of the porin AlgE. Our 2.5 A structure shows AlgK is composed of 9.5 tetratricopeptide-like repeats, and three putative sites of protein-protein interaction have been identified. Bioinformatics analysis suggests that BcsA, PgaA, and PelB, involved in the production and export of cellulose, poly-beta-1,6-N-Acetyl-D-glucosamine, and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together, our data suggest that AlgK plays a role in the assembly of the alginate biosynthetic complex and represents the periplasmic component of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems.

Contribution of the periplasmic chaperone Skp to efficient presentation of the autotransporter IcsA on the surface of Shigella flexneri.

IcsA is an outer membrane protein in the autotransporter family that is required for Shigella flexneri pathogenesis. Following its secretion through the Sec translocon, IcsA is incorporated into the outer membrane in a process that depends on YaeT, a component of an outer membrane beta-barrel insertion machinery. We investigated the role of the periplasmic chaperone Skp in IcsA maturation. Skp is required for the presentation of the mature amino terminus (alpha-domain) of IcsA on the bacterial surface and contributes to cell-to-cell spread of S. flexneri in cell culture. A mutation in skp does not prevent the insertion of the beta-barrel into the outer membrane, suggesting that the primary role of Skp is the folding of the IcsA alpha-domain. In addition, the requirement for skp can be partially bypassed by disrupting icsP, an ortholog of Escherichia coli ompT, which encodes the protease that processes IcsA between the mature amino terminus and the beta-barrel outer membrane anchor. These findings are consistent with a model in which Skp plays a critical role in the chaperoning of the alpha-domain of IcsA during transit through the periplasm.

Porphyromonas gingivalis resistance to polymyxin B is determined by the lipid A 4'-phosphatase, PGN_0524.

AIM: To elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B. METHODOLOGY: A genetic screen of P. gingivalis clones generated by a Tn4400'-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B (PMB, 50 microg x mL(-1)). RESULTS: P. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 microg x mL(-1)). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 microg x mL(-1)). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate. CONCLUSION: The combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization.

Mucoid strains of Pseudomonas aeruginosa that overproduce alginate are associated with chronic pulmonary disease (e.g. cystic fibrosis). Mutants defective in one of several periplasmic proteins (AlgKGX) for alginate secretion release alginate fragments due to the activity of an alginate lyase (AlgL) in the periplasm, which cleaves the newly formed polymers. However, mutants defective in Alg8 or Alg44 did not secrete polymer or alginate fragments, suggesting that both these membrane proteins have a role in the polymerization reaction. A model for the membrane topology of Alg8, a glycosyltransferase (GT), was constructed using PhoA fusions. This provided evidence for a large cytoplasmic loop containing the active domains predicted for beta-GTs such as Alg8 and five transmembrane (TM) domains, one of which resembles a cleavable signal peptide. The C-terminal TM domain of Alg8 was critical for the polymerization reaction in vivo. Alanine substitution mutagenesis showed that all of the predicted active site residues in the widely spaced D, DxD, D, LxxRW motif were required for polymerization activity in vivo, and two of these substitutions also affected Alg8 protein stability. A membrane topology model for Alg44 was also constructed using PhoA fusions, and this showed a central TM domain and predicted an N-terminal TM domain that may be a membrane anchor. An N-terminal PilZ domain in Alg44 for c-di-GMP [bis-(3',5')-cyclic dimeric GMP] binding, which is required for alginate synthesis, was localized to the cytoplasmic loop. The long periplasmic C terminus of Alg44 contains a region similar to membrane fusion proteins (MFPs) of multi-drug efflux systems, which predicts the possibility of its interaction with another protein in this compartment. A Western blot analysis of the outer-membrane porin AlgE showed reduced AlgE levels in the alg44 mutant, whereas expression of Alg44 in trans restored AlgE within the cell. C-terminal truncations of Alg44 as small as 24 amino acids blocked alginate polymerization in vivo, indicating a critical role for the MFP domain. These studies suggest that Alg44 may act as a co-polymerase in concert with Alg8, the major GT, and that both inner-membrane proteins are required in vivo for the polymerization reaction leading to alginate production.