Molecular cloning, sequencing, and characterization of cDNA for sarcotoxin IIA, an inducible antibacterial protein of Sarcophaga peregrina (flesh fly)

A cDNA clone for sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina (flesh fly) larvae [Ando, K., Okada, M., & Natori, S. (1987) Biochemistry 26, 226-230], was isolated and characterized. Sarcotoxin IIA was found to consist of 270 amino acid residues. Northern blot analysis showed that the sarcotoxin IIA gene was activated in response to injury of the body wall of the larvae. The gene was activated for much longer after injection of Escherichia coli into the abdominal cavity of larvae than after injection of saline alone. A common nucleotide sequence for mammalian inflammatory mediator protein cDNAs, TTATTTAT, was found in the 3'-untranslated region of sarcotoxin IIA cDNA, suggesting that this protein plays a role in the inflammatory response of this insect.     

Ionophore activity of sarcotoxin I, a bactericidal protein of Sarcophaga peregrina.

When Escherichia coli was treated with sarcotoxin I, a potent bactericidal protein of Sarcophaga peregrina (fleshfly), K+ inside of the cells leaked out rapidly and the ATP pool of the cells rapidly decreased. These results suggested that the bactericidal effect of sarcotoxin I was due to its ionophore activity, and that it blocked the generation of ATP by inhibiting formation of the proton gradient essential for oxidative phosphorylation. This was confirmed by use of an uncA mutant, which was much less susceptible than the wild-type strain to sarcotoxin I under fixed ionic conditions.     

Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells

Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide.     

Purification of sarcotoxin II, antibacterial proteins of Sarcophaga peregrina (flesh fly) larvae

Three antibacterial proteins with almost identical primary structures termed sarcotoxin IIA, IIB, and IIC were purified to homogeneity from the hemolymph of third instar larvae of Sarcophaga peregrina. The molecular masses of these proteins were about 24,000. These proteins were found to have common antigenicity, and antibody against sarcotoxin IIA cross-reacted with sarcotoxin IIB and IIC. Radioimmunoassay using this antibody showed that these proteins are induced in the hemolymph in response to injury of the larval body wall.     

Interaction between liposomes and sarcotoxin IA, a potent antibacterial protein of Sarcophaga peregrina (flesh fly)

The direct interaction between phospholipids and sarcotoxin IA, a potent bactericidal protein of Sarcophaga peregrina, was studied using authentic sarcotoxin IA, its synthetic derivatives, and various liposomes. Results showed that sarcotoxin IA interacted with liposomes constituted from acidic phospholipids, resulting in the release of glucose trapped in these liposomes. The amidated carboxyl-terminal of this protein was found to be important for this interaction. Liposomes constituted from total phospholipids of Escherichia coli became less susceptible to sarcotoxin IA with an increase in their cholesterol content. Since bacterial membranes do not contain cholesterol, this finding may partly explain the selective toxicity of sarcotoxin I to bacteria.     

Polysaccharides cellulose, poly-beta-1,6-n-acetyl-D-glucosamine, and colanic acid are required for optimal binding of Escherichia coli O157:H7 strains to alfalfa sprouts and K-12 strains to plastic but not for binding to epithelial cells

When Escherichia coli O157:H7 bacteria are added to alfalfa sprouts growing in water, the bacteria bind tightly to the sprouts. In contrast, laboratory K-12 strains of E. coli do not bind to sprouts under similar conditions. The roles of E. coli O157:H7 lipopolysaccharide (LPS), capsular polysaccharide, and exopolysaccharides in binding to sprouts were examined. An LPS mutant had no effect on the binding of the pathogenic strain. Cellulose synthase mutants showed a significant reduction in binding; colanic acid mutants were more severely reduced, and binding by poly-beta-1,6-N-acetylglucosamine (PGA) mutants was barely detectable. The addition of a plasmid carrying a cellulose synthase gene to K-12 strains allowed them to bind to sprouts. A plasmid carrying the Bps biosynthesis genes had only a marginal effect on the binding of K-12 bacteria. However, the introduction of the same plasmid allowed Sinorhizobium meliloti and a nonbinding mutant of Agrobacterium tumefaciens to bind to tomato root segments. These results suggest that although multiple redundant protein adhesins are involved in the binding of E. coli O157:H7 to sprouts, the polysaccharides required for binding are not redundant and each polysaccharide may play a distinct role. PGA, colanic acid, and cellulose were also required for biofilm formation by a K-12 strain on plastic, but not for the binding of E. coli O157:H7 to mammalian cells.     

The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens

The probiotic Escherichia coli strain Nissle 1917 (Mutaflor) of serotype O6:K5:H1 was reported to protect gnotobiotic piglets from infection with Salmonella enterica serovar Typhimurium. An important virulence property of Salmonella is invasion of host epithelial cells. Therefore, we tested for interference of E. coli strain Nissle 1917 with Salmonella invasion of INT407 cells. Simultaneous administration of E. coli strain Nissle 1917 and Salmonella resulted in up to 70% reduction of Salmonella invasion efficiency. Furthermore, invasion of Yersinia enterocolitica, Shigella flexneri, Legionella pneumophila and even of Listeria monocytogenes were inhibited by the probiotic E. coli strain Nissle 1917 without affecting the viability of the invasive bacteria. The observed inhibition of invasion was not due to the production of microcins by the Nissle 1917 strain because its isogenic microcin-negative mutant SK22D was as effective as the parent strain. Reduced invasion rates were also achieved if strain Nissle 1917 was separated from the invasive bacteria as well as from the INT407 monolayer by a membrane non-permeable for bacteria. We conclude E. coli Nissle 1917 to interfere with bacterial invasion of INT407 cells via a secreted component and not relying on direct physical contact with either the invasive bacteria or the epithelial cells.     

Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A(2).

Group IIA secreted phospholipase A(2) (sPLA2) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of recombinant murine and human groups I, II, V, X, and XII sPLA2s on Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. The rank order potency among human sPLA2s against Gram-positive bacteria is group IIA > X > V > XII > IIE > IB, IIF (for murine sPLA2s: IIA > IID > V > IIE > IIC, X > IB, IIF), and only human group XII displays detectable bactericidal activity against the Gram-negative bacterium E. coli. These studies show that highly basic sPLA2s display potent bactericidal activity with the exception of the ability of the acidic human group X sPLA2 to kill Gram-positive bacteria. By studying the Bacillus subtilis and S. aureus bactericidal potencies of a large panel of human group IIA mutants in which basic residues were mutated to acidic residues, it was found that: 1) the overall positive charge of the sPLA2 is the dominant factor in dictating bactericidal potency; 2) basic residues on the putative membrane binding surface of the sPLA2 are modestly more important for bactericidal activity than are other basic residues; 3) relative bactericidal potency tracks well with the ability of these mutants to degrade phospholipids in the bacterial membrane; and 4) exposure of the bacterial membrane of Gram-positive bacteria by disruption of the cell wall dramatically reduces the negative effect of charge reversal mutagenesis on bactericidal potency.     

Factor of salinity and adaptive capacity of recombinant strains of Escherichia coli and Bacillus subtilis

Effect of different concentrations of salts on natural and recombinant strains of Bacillus subtilis and Escherichia coli was studied. The recombinant strain of B. subtilis was found to be more osmotolerant than the wild-type strain of this bacterium, whereas the opposite situation was observed for the recombinant and wild-type strains of E. coli. Some salts exerted a bacteriostatic effect on E. coli and B. subtilis. The adaptive capacity of recombinant strains depended on the number of plasmid copies in the cells. The introduction of recombinant bacteria into model ecosystems resulted in the generation of their variants with increased osmotolerance.     

Novel effects of the prototype translocating Escherichia coli, strain C25 on intestinal epithelial structure and barrier function

Intestinal bacteria play an etiologic role in triggering and perpetuating chronic inflammatory bowel disorders. However, the precise mechanisms whereby the gut microflora influences intestinal cell function remain undefined. Therefore, the effects of the non-pathogenic prototype translocating Escherichia coli, strain C25 on the barrier properties of human T84 and Madine-Darby canine kidney type 1 epithelial cells were examined. T-84 cells were also infected with commensal E. coil, strains F18 and HB101, and enterohaemorrhagic E. coli, serotype O157:H7. Strains F18 and HB101 had no effect on transepithelial electrical resistance (TER) of T84 monolayers. By contrast, epithelial cells infected with strain C25 displayed a time-dependent decrease in TER, preceded by an altered distribution of the cytoskeletal protein alpha-actinin, comparable to infection with E. coli O157:H7. E. coli C25 infection also led to activation of nuclear factor kappaB (NF-kappaB), interleukin-8 secretion and alterations in localization of claudin-1, but not zona occludens-1 or claudin-4, in T84 cells. There were adherent C25 bacteria on the intact apical surface of infected T84 cells, while mitochondria appeared swollen and vacuolated. These novel findings demonstrate the ability of a translocating commensal bacterium to adhere to and modulate intestinal epithelial barrier function and to induce morphological changes in a manner distinct from the known enteric pathogen, E. coli O157:H7.     

Spent media from cultures of environmental isolates of Escherichia coli can suppress the deficiency of biofilm formation under anoxic conditions of laboratory E. coli strains

The prevailing lifestyle of bacteria is sessile and they attach to surfaces in structures known as biofilms. In Escherichia coli, as in many other bacteria, biofilms are formed at the air-liquid interface, suggesting that oxygen has a critical role in the biofilm formation process. It has been reported that anaerobically growing E. coli laboratory strains are unable to form biofilms even after 96 h of incubation on Luria Bertani (LB) medium. After analyzing 22,000 transposon-induced and 26,000 chemically-induced mutants we failed to isolate an E. coli laboratory strain with the ability to form biofilm under anaerobic growth conditions. Notably, seven strains from a collection of E. coli isolated from different hosts and the environment had the ability to form biofilm in the absence of oxygen. Interestingly, spent medium from cultures of one strain, Souza298, can promote biofilm formation of E. coli laboratory strains growing under anaerobic conditions. Our results led us to propose that laboratory E. coli strains do not release (or synthesize) a molecule needed for biofilm formation under anoxic conditions but that they bear all the required machinery needed for this process.     

Saccharomyces cerevisiae cells harboring the gene encoding sarcotoxin IA secrete a peptide that is toxic to plant pathogenic bacteria.

Sarcotoxin IA is a cecropin-type antibacterial protein produced by the flesh fly, Sarcophaga peregrina. Similar to other bactericidal small proteins produced by insects, sarcotoxin IA is released into the hemolymph of larvae and nymphs upon mechanical injury or bacterial infection. The gene (sarco) that encodes this toxin was introduced into Saccharomyces cerevisiae yeast cells and was expressed under a constitutive yeast promoter. The transformed yeast cells were grown in a liquid medium, and a peptide with a similar molecular size to that of the mature sarcotoxin IA was detected in the medium by Western blot analysis. The secreted sarcotoxin-like peptide (SLP) had a potent cytotoxic effect against several bacteria, including plant pathogenic bacteria, similar to the toxic effects of the authentic sarcotoxin IA. Erwinia carotovora was more susceptible to the toxic medium than Pseudomonas solanacearum and Pseudomonas syringae pv. lachrymans. Thus, yeast may be used in the production of such proteins for employment against various bacterial pathogens.     

In vitro antibacterial activity of Lactobacillus helveticus strain KS300 against diarrhoeagenic, uropathogenic and vaginosis-associated bacteria

AIMS: The purpose of this study was to investigate in vitro the antibacterial activity of the Lactobacillus helveticus strain KS300 against vaginosis-associated bacteria including Gardnerella vaginalis and Prevotella bivia, uropathogenic Escherichia coli, and diarrhoeagenic Salmonella enterica serovar Typhimurium. METHODS AND RESULTS: The KS300 strain inhibited the growth of G. vaginalis, P. bivia, S. typhimurium, and pathogenic E. coli. After direct co-culture, data show that the Lactobacillus strain decreased the viability of G. vaginalis, P. bivia, S. typhimurium, and pathogenic E. coli. The adhering KS300 strain inhibited the adhesion of G. vaginalis DSM 4944 and uropathogenic Dr-positive E. coli IH11128 onto HeLa cells. Moreover, the KS300 strain inhibited the internalization of uropathogenic Dr-positive E. coli IH11128 within HeLa cells and S. typhimurium SL1344 within Caco-2/TC7 cells. CONCLUSIONS: The findings demonstrate that L. helveticus strain KS300 is adhesive onto cultured human cells and has antagonistic activities against vaginosis-associated, uropathogenic and diarrhoeagenic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhering L. helveticus strain KS300 is a potential probiotic strain displaying a strain-specific array of in vitro antibacterial activities.     

Spontaneous mutagenesis in exponentially growing and stationary-phase, umuDC-proficient and -deficient, Escherichia coli dnaQ49

Spontaneous mutations arise not only in exponentially growing bacteria but also in non-dividing or slowly dividing stationary-phase cells. In the latter case mutations are called adaptive or stationary-phase mutations. High spontaneous mutability has been observed in temperature sensitive Escherichia coli dnaQ49 strain deficient in 3'-->5' proofreading activity assured by the e subunit of the main replicative polymerase, Pol III. The aim of this study was to evaluate the effects of the dnaQ49 mutation and deletion of the umuDC operon encoding polymerase V (Pol V) on spontaneous mutagenesis in growing and stationary-phase E. coli cells. Using the argE3(OC) -->Arg+ reversion system in the AB1157 strain, we found that the level of growth-dependent and stationary-phase Arg+ revertants was significantly increased in the dnaQ49 mutant at the non-permissive temperature of 37 degrees C. At this temperature, in contrast to cultures grown at 28 degrees C, SOS functions were dramatically increased. Deletion of the umuDC operon in the dnaQ49 strain led to a 10-fold decrease in the level of Arg+ revertants in cultures grown at 37 degrees C and only to a 2-fold decrease in cultures grown at 28 degrees C. Furthermore, in stationary-phase cultures Pol V influenced spontaneous mutagenesis to a much lesser extent than in growing cultures. Our results indicate that the level of Pol III desintegration, dependent on the temperature of incubation, is more critical for spontaneous mutagenesis in stationary-phase dnaQ49 cells than the presence or absence of Pol V.     

HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O-antigenic polysaccharide

The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid-cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the O-repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid-cured derivative strain B171-4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal-like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171-4 was internalized at levels 30-70-fold higher than those of avirulent E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171-4 and an enteroinvasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid-determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell.     

Alteration of Escherichia coli murein during amino acid starvation.

We have studied the mechanisms by which amino acid starvation of Escherichia coli induces resistance against the lytic and bactericidal effects of penicillin. Starvation of E. coli strain W7 of the amino acids lysine or methionine resulted in the rapid development of resistance to autolytic cell wall degradation, which may be effectively triggered in growing bacteria by a number of chemical or physical treatments. The mechanism of this effect in the amino acid-starved cells involved the production of a murein relatively resistant to the hydrolytic action of crude murein hydrolase extracts prepared from normally growing E. coli. Resistance to the autolysins was not due to the covalently linked lipoprotein. Resistance to murein hydrolase developed most rapidly and most extensively in the portion of cell wall synthesized after the onset of amino acid starvation. Lysozymes digests of the autolysin-resistant murein synthesized during the first 10 min of lysine starvation yielded (in addition to the characteristic degradation products) a high-molecular-weight material that was absent from the lysozyme-digests of control cell wall preparations. It is proposed that inhibition of protein synthesis causes a rapid modification of murein structure at the cell wall growth zone in such a manner that attachment of murein hydrolase molecules is inhibited. The mechanism may involve some aspects of the relaxed control system since protection against penicillin-induced lysis developed much slower in amino acid-starved relaxed controlled (relA) cells than in isogenic stringently controlled (relA+) bacteria.     

A Novel Antimicrobial Peptide Derived from the Insect Paederus dermatitis

Much research has been focused on antimicrobial peptides (AMPs) derived from insect immune defense reactions due to their potential in the development of new antibiotics. In this study, a new AMP from the insect Paederus dermatitis, named sarcotoxin Pd was identified and purified using gel filtration and reverse-phase high-performance liquid chromatography. Our results showed that this peptide has broad-spectrum inhibitory effects on examined microbes. Sarcotoxin Pd is composed of 34 amino acids and its molecular weight was estimated to be 3613.26 ± 0.5 Da. Minimum inhibitory concentration (MIC) values of sarcotoxin Pd against Gram-negative bacteria were lower than Gram-positive bacteria and fungi. The identified peptide showed the highest antimicrobial effect against Klebsiella pneumonia and Escherichia coli. This peptide did not reveal significant hemolytic activity against human red blood cells particularly in the range of MIC values. Confirming the potential antimicrobial activities of synthetic peptide, this paper addresses the role of sarcotoxin Pd in the treatment of systemic microbial illnesses.     

Efficient phagocytosis of Klebsiella pneumoniae strains that poorly bind to human polymorphonuclear leukocytes.

The phagocytosis process of unencapsulated MIAT-negative strains that, although binding very poorly to human polymorphonuclear leukocytes (PMN) at 4 degrees C, are efficiently killed by these cells at 37 degrees C, was studied. At 37 degrees C the number of bacteria bound to the PMN external surface was similar to that observed at 4 degrees C (about 100 bacteria/100 PMN after 60 min); on the contrary the number of internalized bacteria was much higher (from 500 bacteria/100 PMN after 60 min). Interactions between phagocytosis-sensitive Klebsiella pneumoniae strains (PSK) and PMN were then compared with those of two isogenic Escherichia coli strains with and without type 1 fimbriae. Whereas PSK strain binding to blocked PMN was very slow and became significant only after 5-6 h, that of phagocytosis-sensitive fimbriated E. coli was rapid and efficient. Phagocytosis-resistant, non fimbriated E. coli strain bound with an efficiency that, within the first 60 min, was not very different from that of the PSK strains. However, longer incubations led to increases in PSK binding, whereas unfimbriated E. coli remained constant. PSK and fimbriated E. coli strains were efficiently internalized and killed, whereas the unfimbriated E. coli strain was not. It is suggested that PMN can phagocytize unopsonized bacteria through two different mechanisms. By one mechanism, observed with the fimbriated E. coli strain, PMN bind many more bacteria than those they can internalize. By the other, observed with PSK strains, PMN bind only the bacteria they can immediately internalize.     

Purification of three antibacterial proteins from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina

Three antibacterial proteins were purified from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). Sequencing studies showed that two of these proteins belong to the sarcotoxin I family, potent antibacterial proteins purified from the hemolymph of Sarcophaga larvae, whereas the other protein, named sapecin, is a new protein consisting of 40 amino acid residues including 6 cysteine residues. Unlike sarcotoxin I, sapecin preferentially represses the growth of various Gram-positive bacteria. The proteins of the sarcotoxin I family produced by this cell line were found to have carboxyl-terminal glycine, whereas sarcotoxin I in the hemolymph has amidated amino acids. This suggests that the embryonic cells lack an enzyme that cleaves off carboxyl-terminal glycine to form a new amidated carboxyl terminus.