Bacterial Outer Membrane Protein OmpX Regulates ß1 Integrin and Epidermal Growth Factor Receptor (EGFR) Involved in Invasion of M-HeLa Cells by Serratia proteamaculans.

Opportunistic pathogen Serratia proteamaculans are able to penetrate the eukaryotic cells. The penetration rate can be regulated by bacterial surface protein OmpX. OmpX family proteins are able to bind to host cell surface to the epidermal growth factor receptor (EGFR) and the extracellular matrix protein fibronectin, whose receptors are in return the α5 ß1 integrins. Here we elucidated the involvement of these host cell proteins in S. proteamaculans invasion. We have shown that, despite the absence of fibronectin contribution to S. proteamaculans invasion, ß1 integrin was directly involved in invasion of M-HeLa cells. Herewith ß1 integrin was not the only receptor that determines sensitivity of host cells to bacterial invasion. Signal transfer from EGFR was also involved in the penetration of these bacteria into M-HeLa cells. However, M-HeLa cells have not been characterized by large number of these receptors. It turned out that S. proteamaculans attachment to the host cell surface resulted in an increment of EGFR and ß1 integrin genes expression. Such gene expression increment also caused Escherichia coli attachment, transformed with a plasmid encoding OmpX from S. proteamaculans. Thus, an OmpX binding to the host cell surface caused an increase in the EGFR and ß1 integrin expression involved in S. proteamaculans invasion.

Bacterial Actin-Specific Endoproteases Grimelysin and Protealysin as Virulence Factors Contributing to the Invasive Activities of Serratia.

The article reviews the discovery, properties and functional activities of new bacterial enzymes, proteases grimelysin (ECP 32) of Serratia grimesii and protealysin of Serratia proteamaculans, characterized by both a highly specific "actinase" activity and their ability to stimulate bacterial invasion. Grimelysin cleaves the only polypeptide bond Gly42-Val43 in actin. This bond is not cleaved by any other proteases and leads to a reversible loss of actin polymerization. Similar properties were characteristic for another bacterial protease, protealysin. These properties made grimelysin and protealysin a unique tool to study the functional properties of actin. Furthermore, bacteria Serratia grimesii and Serratia proteamaculans, producing grimelysin and protealysin, invade eukaryotic cells, and the recombinant Escherichia coli expressing the grimelysin or protealysins gene become invasive. Participation of the cellular c-Src and RhoA/ROCK signaling pathways in the invasion of eukaryotic cells by S. grimesii was shown, and involvement of E-cadherin in the invasion has been suggested. Moreover, membrane vesicles produced by S. grimesii were found to contain grimelysin, penetrate into eukaryotic cells and increase the invasion of bacteria into eukaryotic cells. These data indicate that the protease is a virulence factor, and actin can be a target for the protease upon its translocation into the host cell.

Anti-cancer effects of recombinant arazyme from Serratia Proteomaculans.

PURPOSE: Colorectal cancer is a lethal and prevalent type of cancer in both men and women worldwide, which can develop resistance to cancer chemotherapy. Developing an effective therapeutic agent is the most promising method for this life-threatening disease. The present study aimed to identify, clone, express and purify the recombinant arazyme (r-arazyme) of Serratia proteomaculans and evaluate the antitumor effect of r-arazyme in vitro. METHODS: Bacterial strains and cell line, construction of expression vector and preparation of recombinant protein were prepared and then evaluated by western blot, cell culture, cell viability assay, lactate dehydrogenase release assay, cell apoptosis assay, caspase-3 and -9 activation assay, adhesion assay, matrigel invasion assay and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: R-arazyme caused a great cytotoxic effect against human colorectal adenocarcinoma (HT29) cells in a dose-dependent manner, without any cytotoxic effect on human embryonic kidney cells 293 (HEK 293). In addition, r-arazyme could induce apoptosis in colorectal cancer cell lines via caspase-3 activation and the elevation of the Bax/Bcl-2 ratio. Further, r-arazyme inhibited cancer cells angiogenesis by significantly reducing the expression of angiogenesis-related genes such as VEGF, VEGFR-1, and VEGFR-2. Furthermore, r-arazyme could prevent invasion and adhesion of cancer cells. In general, the results may support the evidence that r-arazyme is a promising therapeutic candidate against cancer. CONCLUSION: R-arazyme may play an important role in developing effective therapies against colorectal adenocarcinoma in humans, which results in reducing the overall morbidity and mortality related to colorectal cancer.

Differential genomic damage in different tumor lines induced by prodigiosin.

Prodigiosin is a secondary metabolite produced by Serratia marcercens. As this pigment is suggested to be a cancer drug, genotoxicity studies are necessary. The aim of the present investigation was to evaluate the genotoxic effects of prodigiosin on tumoral and normal cell lines, NCIH-292, MCF-7 and HL-60. A normal line BGMK was used as control. Genomic damage induced by prodigiosin was observed in all tumor lines as well as the control line. The pigment induced the formation of micronuclei in tumor cells. The present data confirm the antitumor potential of prodigiosin. However, these findings also raise concerns regarding its target-specific action, as genotoxic effects on normal cells also occurred.

Dihydrolipoic but not alpha-lipoic acid affects susceptibility of eukaryotic cells to bacterial invasion.

Sensitivity of eukaryotic cells to facultative pathogens can depend on physiological state of host cells. Previously we have shown that pretreatment of HeLa cells with N-acetylcysteine (NAC) makes the cells 2-3-fold more sensitive to invasion by the wild-type Serratia grimesii and recombinant Escherichia coli expressing gene of actin-specific metalloprotease grimelysin [1]. To evaluate the impact of chemically different antioxidants, in the present work we studied the effects of α-Lipoic acid (LA) and dihydrolipoic acid (DHLA) on efficiency of S. grimesii and recombinant E. coli expressing grimelysin gene to penetrate into HeLa and CaCo cells. Similarly to the effect of NAC, pretreatment of HeLa and CaCo cells with 0.6 or 1.25 mM DHLA increased the entry of grimelysin producing bacteria by a factor of 2.5 and 3 for the wild-type S. grimesii and recombinant E. coli, respectively. In contrast, pretreatment of the cells with 0.6 or 1.25 mM LA did not affect the bacteria uptake. The increased invasion of HeLa and CaCo cells correlated with the enhanced expression of E-cadherin and ß-catenin genes, whereas expression of these genes in the LA-treated cells was not changed. Comparison of these results suggests that it is sulfhydryl group of DHLA that promotes efficient modification of cell properties assisting bacterial uptake. We assume that the NAC- and DHLA-induced stimulation of the E-cadherin-catenin pathway contributes to the increased internalization of the grimelysin producing bacteria within transformed cells.

Virulence factors contributing to invasive activities of Serratia grimesii and Serratia proteamaculans.

Previously, we have shown that facultative pathogens Serratia grimesii and Serratia proteamaculans are capable to invade eukaryotic cells provided that they synthesize intracellular metalloprotease grimelysin or protealysin, respectively (Bozhokina et al. in Cell Biol Int 35(2):111-118, 2011). Noninvasive Escherichia coli transformed with grimelysin or protealysin gene became invasive, indicating that the protease is a virulence factor. Here we elucidated involvement of other virulence factors in the invasion of S. grimesii and S. proteamaculans. Under similar experimental conditions, the amount of S. proteamaculans internalized within human carcinoma HeLa cells was fivefold higher than that of S. grimesii. In accord with this, in S. proteamaculans, high activities of pore-forming hemolysin ShlA and extracellular metalloprotease serralysin were detected. In S. grimesii, activity of toxin ShlA was not detected, and the serralysin activity of the bacterial growth medium was very low. We also show that iron depletion strongly enhanced invasive activity of S. proteamaculans, increasing activities of hemolysin ShlA and serralysin, but did not affect S. grimesii properties. These results show that the invasive activity of S. proteamaculans is maintained, along with protealysin, by hemolysin and serralysin. On the other hand, grimelysin is so far the only known invasion factor of S. grimesii.

[ENTRY OF FACULTATIVE PATHOGEN SERRATIA GRIMESII INTO HELA CELLS. ELECTRON MICROSCOPIC ANALYSIS].

Facultative pathogens Serratia grimesii are able to invade eukaryotic cells where they have been found in vacuoles and free in the cytoplasm (Efremova et al., 2001; Bozhokina et al., 2011). However, efficiency of this invasion is low, and the mechanisms of the invasion related to the initial steps of the process are not known. In the present study, we have increased the invasion efficiency by incubation of HeLa cells with N-acetylcysteine (NAC) preceding the infection. In the NAC-pretreated cells, two modes of S. grimesii to enter HeLa cells were observed. In the most cases, the penetration of S. grimesii into the cell was consistent with the "zipper mechanism", involving specific interaction of bacterial invasin with a host cell surface receptor. However, in some cases, bacteria were trapped by membrane ruffling probably produced by injected bacterial proteins that trigger the bacterial uptake process, as described in the "trigger mechanism". Further elucidation of bacterial and cellular factors involved in the bacteria-host cell interaction should clarify whether two different mechanisms or a predominant one operate during S. grimesii invasion.

Influence of temperature on the physiology and virulence of the insect pathogen Serratia sp. Strain SCBI.

The physiology of a newly recognized Serratia species, termed South African Caenorhabditis briggsae Isolate (SCBI), which is both a nematode mutualist and an insect pathogen, was investigated and compared to that of Serratia marcescens Db11, a broad-host-range pathogen. The two Serratia strains had comparable levels of virulence for Manduca sexta and similar cytotoxic activity patterns, but motility and lipase and hemolytic activities differed significantly between them.

Filamentous actin is a substrate for protealysin, a metalloprotease of invasive Serratia proteamaculans.

Homologous bacterial metalloproteases ECP32/grimelysin from Serratia grimesii and protealysin from Serratia proteamaculans are involved in the invasion of the nonpathogenic bacteria in eukaryotic cells and are suggested to translocate into the cytoplasm [Bozhokina ES et al. (2011) Cell Biol Int35, 111-118]. The proteases have been characterized as actin-hydrolyzing enzymes with a narrow specificity toward intact cell proteins. However, cleavage of filamentous actin (F-actin) (i.e. the main actin species in the cell) and the properties of the cleaved F-actin have not been investigated previously. In the present study, we revealed the presence of protealysin in the cytoplasm of 3T3-SV40 cells infected with S. proteamaculans or recombinant Escherichia coli expressing the protealysin gene. We also show for the first time that purified protealysin and the lysates of the recombinant E. coli producing protealysin cleave 20-40% of F-actin. Cleavage limited predominantly to the bond Gly42-Val43 efficiently increases the steady-state ATPase activity (dynamics) of F-actin. abolishes this effect and promotes the nucleation of protealysin-cleaved Mg-globular-actin even in the absence of 0.1 m KCl, most likely as a result of the stabilization of lateral intermonomer contacts of actin subunits. The results obtained in the present study suggest that F-actin can be a target for protealysin upon its translocation into the host cell.

Probing for actinase activity of protealysin.

The ability of protealysin, a thermolysin-like metallopeptidase from Serratia proteamaculans 94, to cleave actin and matrix metalloprotease MMP2 is reported. In globular actin, protealysin and S. proteamaculans 94 cell extracts are shown to hydrolyze the Gly42-Val43 peptide bond within the DNase-binding loop and the Gly63-Ile64 and Thr66-Ile67 peptide bonds within the nucleotide cleft of the molecule. At enzyme/substrate mass ratio of 1 : 50 and below, a 36 kDa-fragment produced by the cleavage between Gly42 and Val43 was virtually resistant to further breakdown. Judging from the results of zymography, protealysin transforms proMMP2 into a 66 kDa polypeptide characteristic of mature MMP2, indicating that protealysin can activate MMP2. Upon incubation of S. proteamaculans 94 with human larynx carcinoma Hep-2 cells intracellular bacteria were detected in about 10% of Hep-2 cells, this being the first evidence for invasion of eukaryotic cells with bacteria of this species. Thus, S. proteamaculans 94 turned out to be one more bacterial strain in which synthesis of actin-specific metalloprotease is coupled with bacterial invasion. These results are consistent with the idea of the actinase activity of bacterial metalloproteases being a factor that may promote bacterial invasion of eukaryotic cells.

[The story on an intriguing actin-specific protease that turned out to be grimelysin, a member of a respectable family of thermolysin-like metalloproteinases].

The article describes a story of the discovery and properties of bacterial metalloproteinase ECP32 that cleaves actin at the only site between Gly42 and Val43. This site is intensively involved in the monomer-monomer contacts within actin filament. Cleavage with ECP32 results in a reversible loss of actin polymerizability. Therefore ECP cleaved actin has been a unique model to study mechanisms of actin polymerization and the filament dynamics. and the effects of actin-binding proteins on these processes, as well as to reveal allosteric effects in actin molecule and to determine three-dimensional actin structure that was previously determined only for the actin-ligand complexes. Furthermore, the non-pathogenic bacteria synthesizing ECP32 were shown to penetrate in eukaryotic cells rearranging their cytoskeleton. Biochemical analysis using the Vitek-2 system and sequencing of the 16S rRNA gene reidentified the ECP32-producing strain, previously identified as E. coli, as Serratia grimesii. Bacteria of a reference strain S. grimesii were found to express the gene of metalloprotease that cleaves actin similarly to ECP32. The gene was cloned, sequenced and expressed in E. coli. The protein encoded by this gene was named grimelysin. Grimelysin shared essential characteristics of ECP32: molecular weight, limited actin proteolysis, inhibition by chelating agents, cleavage site, and the N-terminal amino acids of the active enzyme published for ECP32. These data show that grimelysin and ECP32 seem to be the same protein. As it was demonstrated by confocal microscopy the bacteria capable of synthesizing natural or recombinant grimelysin acquire invasive phenotype. The data described here allow us to suggest that actin may be a target protein which proteolysis promotes bacterial invasion.

The characterization of upper-room ultraviolet germicidal irradiation in inactivating airborne microorganisms.

In this study, we explored the efficacy of upper-room ultraviolet germicidal irradiation (UVGI) in reducing the concentration of Serratia marcescens and Mycobacterium bovis bacille Calmette-Guérin (BCG) aerosols in enclosed places. We constructed a facility (4.5 m x 3 m x 2.9 m) in which both ceiling- and wall-mounted UV fixtures (UV output: 10W and 5W respectively) were installed. The use of ceiling- and wall-mounted UV fixtures (total UV output: 15W) without mixing fan reduced the concentration of S. marcescens aerosols by 46% (range: 22-80%) at 2 air changes per hour (ACH) and 53% (range: 40-68%) at 6 ACH. The use of ceiling- and wall-mounted UV fixtures with mixing fan increased the UV effectiveness in inactivating S. marcescens aerosols to 62% (range: 50-78%) at 2 ACH and to 86% (81-89%) at 6 ACH. For BCG aerosols, UV effectiveness in inactivating BCG aerosols at 6 ACH were 52% (range: 11-69%) by ceiling-mounted UV fixture only (total UV output: 10W) and 64% (51-83%) by both ceiling- and wall-mounted UV fixtures (total UV output: 15W). Our results indicated that the equivalent ventilation rate attributable to upper-room UVGI for BCG aerosols ranged from 1 ACH to 22 ACH for ceiling-mounted UV fixtures and from 6.4 ACH to 28.5 ACH for ceiling- and wall-mounted UV fixtures. Both generalized linear and generalized additive models were fitted to all our data. The regression results indicated that the number of UV fixtures, use of mixing fan, and air exchange rate significantly affected UV effectiveness (p < 0.01, 0.01, 0.01 respectively). However, the strain difference (S. marcescens vs. BCG) appeared less important in UV effectiveness (p = 0.26). Our results also indicated that UV effectiveness increased at higher temperature ((italic)p(/italic) < 0.01), lower dry-bulb temperature ((italic)p(/italic) = 0.21), and colder air from a supply grill located near the ceiling (p = 0.22).

Fatal reaction to transfusion of red-cell concentrate contaminated with Serratia liquefaciens.

A 60-year-old woman undergoing surgery died from endotoxic shock and DIC after receiving a 19-day-old unit of optimal additive red-cell concentrate found contaminated with Serratia liquefaciens. No source of contamination could be found. This normally free-living organism is usually of low pathogenicity. It is a very unusual contaminant of stored donated blood, although it appears to be on the increase. When transfused, blood contaminated with S. liquefaciens always causes severe morbidity and is associated with a high death rate. This is the fifth report in the English literature.

Serratia rubidaea as an invasive pathogen.

Serratia rubidaea biotype 1 was isolated from the bile and blood of a patient with a bile tract carcinoma obstructing the common bile duct and who underwent invasive procedures. The infection was cleared after adequate treatment with antibiotics.

Report of six cases of human infection by Serratia plymuthica.

Serratia plymuthica is an uncommon cause of human infection. Only one case of chronic osteomyelitis and two cases of sepsis secondary to central venous catheter infection have been documented. We report the isolation of S. plymuthica from six patients. The organism was recovered from blood cultures in three cases in which the patients had lymphoblastic leukemia, lymphoma, or stroke. Two isolates were recovered from exudates (following knee and abdominal surgery). In the last case, the organism was isolated from the peritoneal fluid of a patient with cholecystitis. The infection was considered nosocomial in five cases and community acquired in the other.

Case report and review of septicemia due to Serratia ficaria.

Serratia ficaria was first described in 1979 as part of the fig tree ecosystem (P.A.D. Grimont, F. Grimont, and M. P. Starr, Curr. Microbiol. 2:277-282, 1979). Since then, it has been isolated from clinical specimens from a few human patients (C. Bollet, J. Freney, P. de Micco, F. Grimont, and P.A.D. Grimont, Méd. Mal. Infect. 20:97-100, 1990; J.A. Brouillard, W. Hansen, and A. Compere, J. Clin. Microbiol. 19:902-904, 1984; H. Darbas, H. Jean-Pierre, G. Boyer, and M. Riviere, Méd. Mal. Infect. 23:269-270, 1993; V.J. Gill, J.J. Farmer, III, P.A.D. Grimont, M.A. Asbury, and C.L. McIntosh, J. Clin. Microbiol. 14:234-236, 1981; F.D. Pien and J.J. Farmer III, South. Med. J. 76:1591-1592, 1983; C. Richard, J. de Coquet, and C. Suc, Méd. Mal. Infect. 19:45-47, 1989), but the pathogenicity of S. ficaria was always questionable. We are reporting the case of an aged cancer patient who developed S. ficaria septicemia. The habitat of this organism and its potential role as a pathogen are discussed.

Microorganismos patogenos de hallazgo poco comun en infectologia / Pathogenic microorganisms not frecuently found in infectology

Se estudiaron los microorganismos de identificacion dificil referidos al laboratorio de bacteriologia del Instituto Nacional de Salud por 5 Instituciones. En 70 casos fue posible obtener una identificacion completa y responsabilizar al microorganismo aislado como agente etiologico de un cuadro clinico. Se trataba de 28 cuadros de septicemia, 26 de meningitis, 4 respiratorios, 7 genitourinarios y 6 varios que incluyeron heridas, abcesos, y conjuntivitis. Los identificados fueron: Serratia en el 38%, Acinetobacter en el 21.4%, Moraxella en el 10%, Alcaligenes en el 8.7%, Aeromonas en el 7.2%, Listeria en el 5.7%, Estreptobacilos en el 4.3% Corynebacterium en el 4.3%, Achromabacter, Cardiobacterium y los grupos M3 y M4 aparece cada uno con el 1.4% constituyendose en verdaderas curiosidades biologicas

Gram-negative bacillary pneumonia in the compromised host.

The clinical and radiological characteristics of 217 consecutive episodes of gram-negative bacillary pneumonia occurring in 189 adult cancer patients between November 1968 and December 1974 were analyzed. The majority of patients had acute leukemia (54%). Fever larger than or equal to 101 degrees F was the single most common symptom and sign of the presence of infection (90%). Next in frequency were crepitant rales (65%), cough (41%), dyspnea (19%) and chest pain (18%). Radiographic evidence of pneumonia was found in 83% of cases and it consisted mainly of alveolar infiltrates involving both lung fields and predominantly the bases. Up to one-third of the patients had normal chestx-ray examinations at the onset of infection, though they subsequently became abnormal in 42% of them. The majority of patients (81%) whose initial chest x-rays did not reveal the presence of pneumonia were neutropenic (less than 1000 circumlating neutrophils/mm3). Klebsiella sp. and Pseudomonas sp. were the most common infecting organisms. The overall cure rate was 61%; 70% for Klebsiella sp. infections and 64% for Pseudomonas sp. infections. Pulmonary abscesses occurred in 14% of the cases. Cures were related to the antibiotic sensitivity of the infecting organisms and to the number of circulating neutrophils during the period of infection. Best results were obtained with the administration of gentamicin, the newer aminoglycoside antibiotic sisomicin, tobramycin and amikacin, or the combination of gentamicin with carbenicillin or with cephalosporins. Early and vigorous therapy of gram-negative bacillary pneumonia with appropriate antibiotics has improved the prognosis of this infection at our institution.