Agnieszka Maszewska

Proteus sp. – an opportunistic bacterial pathogen – classification, swarming growth, clinical significance and virulence factors

ABSTRACT The genus Proteus belongs to the Enterobacteriaceae family, where it is placed in the tribe Proteeae, together with the genera Morganella and Providencia. Currently, the genus Proteus consists of five species: P. mirabilis, P. vulgaris, P. penneri, P. hauseri and P. myxofaciens, as well as three unnamed Proteus genomospecies. The most defining characteristic of Proteus bacteria is a swarming phenomenon, a multicellular differentiation process of short rods to elongated swarmer cells. It allows population of bacteria to migrate on solid surface. Proteus bacteria inhabit the environment and are also present in the intestines of humans and animals. These microorganisms under favorable conditions cause a number of infections including urinary tract infections (UTIs), wound infections, meningitis in neonates or infants and rheumatoid arthritis. Therefore, Proteus is known as a bacterial opportunistic pathogen. It causes complicated UTIs with a higher frequency, compared to other uropathogens. Proteus infections are accompanied by a formation of urinary stones, containing struvite and carbonate apatite. The virulence of Proteus rods has been related to several factors including fimbriae, flagella, enzymes (urease - hydrolyzing urea to CO2 and NH3, proteases degrading antibodies, tissue matrix proteins and proteins of the complement system), iron acqusition systems and toxins: hemolysins, Proteus toxin agglutinin (Pta), as well as an endotoxin - lipopolysaccharide (LPS). Proteus rods form biofilm, particularly on the surface of urinary catheters, which can lead to serious consequences for patients. In this review we present factors involved in the regulation of swarming phenomenon, discuss the role of particular pathogenic features of Proteus spp., and characterize biofilm formation by these bacteria.

Structure of the O-polysaccharide of Providencia alcalifaciens O21 containing 3-formamido-3,6-dideoxy-d-galactose

The O-polysaccharide (O-antigen) of Providencia alcalifaciens O21 was obtained by mild acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy. It was found that the polysaccharide is built up of branched pentasaccharide repeating units with a terminal residue of 3-formamido-3,6-dideoxy-D-galactose (D-Fuc3NFo) and has the following structure: [structure: see text]. Anti-P. alcalifaciens O21 serum cross-reacted with the O-antigen of Proteus vulgaris O47, which contains a GalNAc trisaccharide similar to that present in the P. alcalifaciens O21 O-polysaccharide.

Structure of the O-polysaccharide of Providencia alcalifaciens O8 containing (2S,4R)-2,4-dihydroxypentanoic acid, a new non-sugar component of bacterial glycans.

A glycerol teichoic acid-like O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O8 and studied by chemical methods and NMR spectroscopy, including 2D ROESY, {(1)H,(13)C} HSQC, and HMQC-TOCSY experiments. It was found that the compound contains a new component of bacterial lipopolysaccharides: ether-linked (2S,4R)-2,4-dihydroxypentanoic acid (Dhpa), which was identified by NMR spectroscopy. The following structure of the repeating unit of the polysaccharide was established: [structure: see text]

The amino acid sequences and activities of synergistic hemolysins from Staphylococcus cohnii

Staphylococcus cohnii ssp. cohnii and S. cohnii ssp. urealyticus are a coagulase-negative staphylococci considered for a long time as unable to cause infections. This situation changed recently and pathogenic strains of these bacteria were isolated from hospital environments, patients and medical staff. Most of the isolated strains were resistant to many antibiotics. The present work describes isolation and characterization of several synergistic peptide hemolysins produced by these bacteria and acting as virulence factors responsible for hemolytic and cytotoxic activities. Amino acid sequences of respective hemolysins from S. cohnii ssp. cohnii (named as H1C, H2C and H3C) and S. cohnii ssp. urealyticus (H1U, H2U and H3U) were identical. Peptides H1 and H3 possessed significant amino acid homology to three synergistic hemolysins secreted by Staphylococcus lugdunensis and to putative antibacterial peptide produced by Staphylococcus saprophyticus ssp. saprophyticus. On the other hand, hemolysin H2 had a unique sequence. All isolated peptides lysed red cells from different mammalian species and exerted a cytotoxic effect on human fibroblasts.

Enterocyte-like Caco-2 cells as a model for in vitro studies of diarrhoeagenic Providencia alcalifaciens invasion.

The entry of Providencia alcalifaciens into the enterocyte-like cell line Caco-2 compared to HEp-2 was studied. Of the 22 P. alcalifaciens strains, 13 and 21 were invasive for Caco-2 and HEp-2 cells, respectively. In contrast to HEp-2 cells, P. alcalifaciens was internalised by Caco-2 cells via receptor-mediated endocytosis. Tyrosine kinases play an important role in P. alcalifaciens uptake, also microfilaments and microtubules are engaged in this process. Inhibition of endosome acidification by ammonium chloride did not seem to have any significant effect on P. alcalifaciens invasion. Similarly to Shigella flexnerii, the invasion of Caco-2 cells by these bacteria occurred more effectively through the basolateral pole than through the apical surface of these cells. Plasmid DNA analysis showed the presence of plasmids of 5-172 kb in 13 strains regardless of their invasive ability. The presence of extracellular bacterial protein, most likely a kind of an invasin, is required for the invasion of Caco-2 and HEp-2 cells.

Structure of the O-polysaccharide of Providencia alcalifaciens O35 containing an N-[(S)-1-carboxyethyl]-L-alanine (alanopine) derivative of 4-amino-4,6-dideoxyglucose.

The O-polysaccharide of Providencia alcalifaciens O35 was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(13)C HMBC, and NOESY experiments in D2O and, to detect correlations for NH protons, in a 9:1 H2O/D2O mixture. A unique N-(1-carboxyethyl)alanine (alanopine, Alo) derivative of 4-amino-4,6-dideoxyglucose (Qui4N) was identified as the polysaccharide component. Alanopine was isolated by solvolysis of the polysaccharide with triflic acid followed by acid hydrolysis, and its (2S,4S)-configuration was determined by the specific optical rotation. The following structure of the O-polysaccharide was established (the d configuration of Qui4N was ascribed tentatively): [structure: see text].

Novel tetrahydroacridine and cyclopentaquinoline derivatives with fluorobenzoic acid moiety induce cell cycle arrest and apoptosis in lung cancer cells by activation of DNA damage signaling

Lung cancer is still the leading cause of cancer-related death worldwide, indicating a necessity to develop more effective therapy. Acridine derivatives are potential anticancer agents due to their ability to intercalate DNA as well as inhibit enzymes involved in replication and transcription. Recently, we have evaluated anticancer activity of 32 novel acridine-based compounds. We found that the most effective were tetrahydroacridine and cyclopentaquinoline derivatives with fluorobenzoic acid containing eight and nine carbon atoms in the aliphatic chain. The aim of this study was to determine the molecular mechanisms of compounds-induced cell cycle arrest and apoptosis in human lung adenocarcinoma cells. All compounds activated Ataxia telangiectasia mutated kinase and phosphorylated histone H2A.X at Ser139 indicating DNA damage. Treatment of cells with the compounds increased phosphorylation and accumulation of p53 that regulate cell cycle as well as apoptosis. All compounds induced G0/1 cell cycle arrest by phosphorylation of cyclin-dependent kinase 2 at Tyr15 resulting in attenuation of the kinase activity. In addition, cyclopentaquinoline derivatives induced expression of cyclin-dependent kinase 2 inhibitor, p21; however, tetrahydroacridine derivatives had no significant effect on p21. Moreover, all compounds decreased the mitochondrial membrane potential accompanied by increased expression of Bax and down-regulation of Bcl-2, suggesting activation of the mitochondrial pathway. All compounds also significantly attenuated the migration rates of lung cancer cells. Collectively, our findings suggest a central role of activation of DNA damage signaling in response to new acridine derivatives treatment to induce cell cycle arrest and apoptosis in cancer cells and provide support for their further development as potential drug candidates.

Synergistic hemolysins of coagulase-negative staphylococci (CoNS).

A total of 104 coagulase negative staphylococci, belonging to S. capitis, S. hominis, S. haemolyticus and S. warneri, originating from the collection of the Department of Pharmaceutical Microbiology (ZMF), Medical University of Lodz, Poland, were tested for their synergistic hemolytic activity. 83% of strains produced δ-hemolysin, however, the percentage of positive strains of S. haemolyticus, S. warneri, S. capitis and S. hominis was different - 98%, 78%, 75% and 68%, respectively. Highly pure hemolysins were obtained from culture supernatants by protein precipitation with ammonium sulphate (0-70% of saturation) and extraction by using a mixture of organic solvents. The purity and molecular mass of hemolysins was determined by TRIS/Tricine PAGE. All CoNS hemolysins were small peptides with a molar mass of about 3.5 kDa; they possessed cytotoxic activity against the line of human foreskin fibroblasts ATCC Hs27 and lysed red cells from different mammalian species, however, the highest activity was observed when guinea pig, dog and human red blood cells were used. The cytotoxic effect on fibroblasts occurred within 30 minutes. The S. cohnii ssp. urealyticus strain was used as a control. The antimicrobial activity was examined using hemolysins of S. capitis, S. hominis, S. cohnii ssp. cohnii and S. cohnii ssp. urealyticus. Hemolysins of the two S. cohnii subspecies did not demonstrate antimicrobial activity. Cytolysins of S. capitis and S. hominis had a very narrow spectrum of action; out of 37 examined strains, the growth of only Micrococcus luteus, Corynebacterium diphtheriae and Pasteurella multocida was inhibited.