Publications by authors named "Orietta Massidda"

24 Publications

  • Page 1 of 1

Impact of Prior Antibiotic Use in Primary Care on Resistance to Third Generation Cephalosporins: A Case-Control Study.

Antibiotics (Basel) 2021 Apr 16;10(4). Epub 2021 Apr 16.

Department of Medical Sciences and Public Health, Faculty of Medicine and Surgery, University of Cagliari, 09124 Cagliari, Italy.

Research is lacking on the reversibility of antimicrobial resistance (AMR). Thus, we aimed to determine the influence of previous antibiotic use on the development and decay over time of third generation cephalosporin (3GC)-resistance of . Using the database of hospital laboratories of the Autonomous Province of Bolzano/Bozen (Italy), anonymously linked to the database of outpatient pharmaceutical prescriptions and the hospital discharge record database, this matched case-control study was conducted including as cases all those who have had a positive culture from any site for 3GC resistant (3GCREC) during a 2016 hospital stay. Data were analyzed by conditional logistic regression. 244 cases were matched to 1553 controls by the date of the first isolate. Male sex (OR 1.49, 95% CI 1.10-2.01), older age (OR 1.11, 95% CI 1.02-1.21), the number of different antibiotics taken in the previous five years (OR 1.20, 95% CI 1.08-1.33), at least one antibiotic prescription in the previous year (OR 1.92, 95% CI 1.36-2.71), and the diagnosis of diabetes (OR 1.57, 95% CI 1.08-2.30) were independent risk factors for 3GCREC colonization/infection. Patients who last received an antibiotic prescription two years or three to five years before hospitalization showed non-significant differences with controls (OR 0.97, 95% CI 0.68-1.38 and OR 0.85, 95% CI 0.59-1.24), compared to an OR of 1.92 (95% CI 1.36-2.71) in those receiving antibiotics in the year preceding hospitalization. The effect of previous antibiotic use on 3GC-resistance of is highest after greater cumulative exposure to any antibiotic as well as to 3GCs and in the first 12 months after antibiotics are taken and then decreases progressively.
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http://dx.doi.org/10.3390/antibiotics10040451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073604PMC
April 2021

The Cell Wall of .

Microbiol Spectr 2019 05;7(3)

The Rockefeller University, New York, NY.

has a complex cell wall that plays key roles in cell shape maintenance, growth and cell division, and interactions with components of the human host. The peptidoglycan has a heterogeneous composition with more than 50 subunits (muropeptides)-products of several peptidoglycan-modifying enzymes. The amidation of glutamate residues in the stem peptide is needed for efficient peptide cross-linking, and peptides with a dipeptide branch prevail in some beta-lactam-resistant strains. The glycan strands are modified by deacetylation of -acetylglucosamine residues and -acetylation of -acetylmuramic acid residues, and both modifications contribute to pneumococcal resistance to lysozyme. The glycan strands carry covalently attached wall teichoic acid and capsular polysaccharide. Pneumococci are unique in that the wall teichoic acid and lipoteichoic acid contain the same unusually complex repeating units decorated with phosphoryl choline residues, which anchor the choline-binding proteins. The structures of lipoteichoic acid and the attachment site of wall teichoic acid to peptidoglycan have recently been revised. During growth, pneumococci assemble their cell walls at midcell in coordinated rounds of cell elongation and division, leading to the typical ovococcal cell shape. Cell wall growth depends on the cytoskeletal FtsA and FtsZ proteins and is regulated by several morphogenesis proteins that also show patterns of dynamic localization at midcell. Some of the key regulators are phosphorylated by StkP and dephosphorylated by PhpP to facilitate robust selection of the division site and plane and to maintain cell shape.
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http://dx.doi.org/10.1128/microbiolspec.GPP3-0018-2018DOI Listing
May 2019

The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes.

Nat Commun 2019 01 16;10(1):261. Epub 2019 Jan 16.

Institute for Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK.

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here, we describe critical structural features for the interaction of GpsB with peptidoglycan synthases from three bacterial species (Bacillus subtilis, Listeria monocytogenes and Streptococcus pneumoniae) and suggest their importance for cell wall growth and viability in L. monocytogenes and S. pneumoniae. We use these structural motifs to identify novel partners of GpsB in B. subtilis and extend the members of the GpsB interactome in all three bacterial species. Our results support that GpsB functions as an adaptor protein that mediates the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner.
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http://dx.doi.org/10.1038/s41467-018-08056-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335420PMC
January 2019

Absence of the KhpA and KhpB (JAG/EloR) RNA-binding proteins suppresses the requirement for PBP2b by overproduction of FtsA in Streptococcus pneumoniae D39.

Mol Microbiol 2017 Dec 2;106(5):793-814. Epub 2017 Nov 2.

Department of Biology, Indiana University Bloomington (IUB), Bloomington, IN 47405, USA.

Suppressor mutations were isolated that obviate the requirement for essential PBP2b in peripheral elongation of peptidoglycan from the midcells of dividing Streptococcus pneumoniae D39 background cells. One suppressor was in a gene encoding a single KH-domain protein (KhpA). ΔkhpA suppresses deletions in most, but not all (mltG), genes involved in peripheral PG synthesis and in the gpsB regulatory gene. ΔkhpA mutations reduce growth rate, decrease cell size, minimally affect shape and induce expression of the WalRK cell-wall stress regulon. Reciprocal co-immunoprecipitations show that KhpA forms a complex in cells with another KH-domain protein (KhpB/JAG/EloR). ΔkhpA and ΔkhpB mutants phenocopy each other exactly, consistent with a direct interaction. RNA-immunoprecipitation showed that KhpA/KhpB bind an overlapping set of RNAs in cells. Phosphorylation of KhpB reported previously does not affect KhpB function in the D39 progenitor background. A chromosome duplication implicated FtsA overproduction in Δpbp2b suppression. We show that cellular FtsA concentration is negatively regulated by KhpA/B at the post-transcriptional level and that FtsA overproduction is necessary and sufficient for suppression of Δpbp2b. However, increased FtsA only partially accounts for the phenotypes of ΔkhpA mutants. Together, these results suggest that multimeric KhpA/B may function as a pleiotropic RNA chaperone controlling pneumococcal cell division.
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http://dx.doi.org/10.1111/mmi.13847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719866PMC
December 2017

Suppression and synthetic-lethal genetic relationships of ΔgpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin-binding protein interactions in Streptococcus pneumoniae D39.

Mol Microbiol 2017 03 7;103(6):931-957. Epub 2017 Feb 7.

Department of Biology, Indiana University Bloomington, Bloomington, IN 47405, USA.

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side-wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division-protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co-IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP-PBP2x.
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http://dx.doi.org/10.1111/mmi.13613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5344783PMC
March 2017

Roles of the Essential Protein FtsA in Cell Growth and Division in Streptococcus pneumoniae.

J Bacteriol 2017 Feb 12;199(3). Epub 2017 Jan 12.

Dipartimento di Scienze Chirurgiche, Università di Cagliari, Cagliari, Italy

is an ovoid-shaped Gram-positive bacterium that grows by carrying out peripheral and septal peptidoglycan (PG) synthesis, analogous to model bacilli, such as and In the model bacilli, FtsZ and FtsA proteins assemble into a ring at midcell and are dedicated to septal PG synthesis but not peripheral PG synthesis; hence, inactivation of FtsZ or FtsA results in long filamentous cells unable to divide. Here, we demonstrate that FtsA and FtsZ colocalize at midcell in and that partial depletion of FtsA perturbs septum synthesis, resulting in elongated cells with multiple FtsZ rings that fail to complete septation. Unexpectedly, complete depletion of FtsA resulted in the delocalization of FtsZ rings and ultimately cell ballooning and lysis. In contrast, depletion or deletion of and , which in are synthetically lethal with , resulted in enlarged and elongated cells with multiple FtsZ rings, with deletion of mimicking partial depletion of FtsA. Notably, cell ballooning was not observed, consistent with later recruitment of these proteins to midcell after Z-ring assembly. The overproduction of FtsA stimulates septation and suppresses the cell division defects caused by the deletion of and under some conditions, supporting the notion that FtsA shares overlapping functions with GpsB and SepF at later steps in the division process. Our results indicate that, in , both GpsB and SepF are involved in septal PG synthesis, whereas FtsA and FtsZ coordinate both peripheral and septal PG synthesis and are codependent for localization at midcell. (pneumococcus) is a clinically important human pathogen for which more therapies against unexploited essential targets, like cell growth and division proteins, are needed. Pneumococcus is an ovoid-shaped Gram-positive bacterium with cell growth and division properties that have important distinctions from those of rod-shaped bacteria. Gaining insights into these processes can thus provide valuable information to develop novel antimicrobials. Whereas rods use distinctly localized protein machines at different cellular locations to synthesize peripheral and septal peptidoglycans, we present evidence that organizes these two machines at a single location in the middle of dividing cells. Here, we focus on the properties of the actin-like protein FtsA as an essential orchestrator of peripheral and septal growth in this bacterium.
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http://dx.doi.org/10.1128/JB.00608-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5237122PMC
February 2017

Helicobacter pylori: enemy, commensal or, sometimes, friend?

J Infect Dev Ctries 2015 Jul 4;9(6):674-8. Epub 2015 Jul 4.

Institute for Genetics and Biomecial Research, National Research Council, Cagliari, Italy.

Helicobacter pylori is a Gram-negative ε-proteobacterium that colonizes about 50% of humans. Some pertinent characteristics are that it can survive the acid of the stomach, produces urease to neutralize it and is motile due to apical flagella. Not surprisingly given its wide distribution, it has long colonized mankind and its genome encodes many features that allows this. Consequently, it frequently has a persistent lifelong association with humans and, differently from most pathogens that are transmitted horizontally, it is preferentially transmitted vertically, often from mother to child. A variety of genes and polymorphisms, both in H pylori and in humans, mediate the complex host-bacterium relationship, and can also determine if and what pathologies will be triggered by the species. H. pylori is naturally transformable, very recombinogenic and has a high mutation rate. Microbiota studies of the stomach have shown it to be an important species with a potentially regulatory role for the gastric microbial community. Likewise, epidemiological work has suggested that, while it clearly increases the risk of peptic ulcers and gastric cancer in some populations, it is also associated with lower risk of esophageal cancer and several other important pathologies. More recently, antibacterial resistant strains have been isolated, posing a problem for public health officials who called for its eradication. Hence, study of H. pylori and how it interacts with us can help revealing mutualistic or pathogenic interactions and the immune response in the digestive niche.
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http://dx.doi.org/10.3855/jidc.7186DOI Listing
July 2015

LocZ is a new cell division protein involved in proper septum placement in Streptococcus pneumoniae.

mBio 2014 Dec 30;6(1):e01700-14. Epub 2014 Dec 30.

Cell and Molecular Microbiology Division, Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic.

Unlabelled: How bacteria control proper septum placement at midcell, to guarantee the generation of identical daughter cells, is still largely unknown. Although different systems involved in the selection of the division site have been described in selected species, these do not appear to be widely conserved. Here, we report that LocZ (Spr0334), a newly identified cell division protein, is involved in proper septum placement in Streptococcus pneumoniae. We show that locZ is not essential but that its deletion results in cell division defects and shape deformation, causing cells to divide asymmetrically and generate unequally sized, occasionally anucleated, daughter cells. LocZ has a unique localization profile. It arrives early at midcell, before FtsZ and FtsA, and leaves the septum early, apparently moving along with the equatorial rings that mark the future division sites. Consistently, cells lacking LocZ also show misplacement of the Z-ring, suggesting that it could act as a positive regulator to determine septum placement. LocZ was identified as a substrate of the Ser/Thr protein kinase StkP, which regulates cell division in S. pneumoniae. Interestingly, homologues of LocZ are found only in streptococci, lactococci, and enterococci, indicating that this close phylogenetically related group of bacteria evolved a specific solution to spatially regulate cell division.

Importance: Bacterial cell division is a highly ordered process regulated in time and space. Recently, we reported that the Ser/Thr protein kinase StkP regulates cell division in Streptococcus pneumoniae, through phosphorylation of several key proteins. Here, we characterized one of the StkP substrates, Spr0334, which we named LocZ. We show that LocZ is a new cell division protein important for proper septum placement and likely functions as a marker of the cell division site. Consistently, LocZ supports proper Z-ring positioning at midcell. LocZ is conserved only among streptococci, lactococci, and enterococci, which lack homologues of the Min and nucleoid occlusion effectors, indicating that these bacteria adapted a unique mechanism to find their middle, reflecting their specific shape and symmetry.
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http://dx.doi.org/10.1128/mBio.01700-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281919PMC
December 2014

Hard tissue response to argon plasma cleaning/sterilisation of customised titanium abutments versus 5-second steam cleaning: results of a 2-year post-loading follow-up from an explanatory randomised controlled trial in periodontally healthy patients.

Eur J Oral Implantol 2013 ;6(3):251-60

Purpose: The aim of this triple-blinded randomised controlled trial was to test if argon plasma cleaning/ sterilisation of customised abutments can affect peri-implant marginal bone levels when compared to 5 seconds of steam cleaning.

Materials And Methods: A total of 20 consecutive periodontally healthy patients requiring single implant-supported restorations in the maxillary premolar or anterior area were selected. All patients received a single implant. At abutment connection, customised abutments were randomly allocated to control (subjected only to usually adopted steam cleaning, CG) and test groups (subjected to plasma cleaning/sterilisation, TG). Abutments were screwed in at 32 Ncm, provisional restorations adapted and periapical radiographs were taken using customised film holders. Two weeks later, definitive restorations were placed. Patients were followed-up for 2 years post-loading. Outcome measures were implant/crown success, complications, periapical marginal bone level changes on periapical standardised radiographs, and microbiological analyses of the abutments after customisation and cleaning procedures but before connection. Comparisons between groups were performed by independent sample t tests (significance threshold of P ≤ 0.05).

Results: No patient dropped out 2 years after loading. The presence of bacterial growth (staphylococci, including Staphylococcus aureus) was observed only on the CG abutments. No implant failed and no complications occurred. After 2 years of prosthetic loading, radiographic analysis revealed a statistically significantly higher mean bone loss for the CG group (mean difference 0.4 mm; 95% CI 0.08-0.73; P = 0.018).

Conclusions: This study suggests that removal of contaminants from titanium abutments using plasma of argon can allow for better bone level maintenance when compared to 5-second steam cleaning of titanium abutments. It is therefore important to use cleaned and sterilised customised abutments in patients.
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January 2014

From models to pathogens: how much have we learned about Streptococcus pneumoniae cell division?

Environ Microbiol 2013 Dec 15;15(12):3133-57. Epub 2013 Jul 15.

Department of Surgical Sciences, University of Cagliari, Via Porcell, 4, 09100, Cagliari, Italy.

Streptococcus pneumoniae is an oval-shaped Gram-positive coccus that lives in intimate association with its human host, both as a commensal and pathogen. The seriousness of pneumococcal infections and the spread of multi-drug resistant strains call for new lines of intervention. Bacterial cell division is an attractive target to develop antimicrobial drugs. This review discusses the recent advances in understanding S. pneumoniae growth and division, in comparison with the best studied rod-shaped models, Escherichia coli and Bacillus subtilis. To maintain their shape, these bacteria propagate by peripheral and septal peptidoglycan synthesis, involving proteins that assemble into distinct complexes called the elongasome and the divisome, respectively. Many of these proteins are conserved in S. pneumoniae, supporting the notion that the ovococcal shape is also achieved by rounds of elongation and division. Importantly, S. pneumoniae and close relatives with similar morphology differ in several aspects from the model rods. Overall, the data support a model in which a single large machinery, containing both the peripheral and septal peptidoglycan synthesis complexes, assembles at midcell and governs growth and division. The mechanisms generating the ovococcal or coccal shape in lactic-acid bacteria have likely evolved by gene reduction from a rod-shaped ancestor of the same group.
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http://dx.doi.org/10.1111/1462-2920.12189DOI Listing
December 2013

Streptococcus pneumoniae transposon Tn1545/Tn6003 changes to Tn6002 due to spontaneous excision in circular form of the erm(B)- and aphA3-containing macrolide-aminoglycoside-streptothricin (MAS) element.

Antimicrob Agents Chemother 2012 Nov 13;56(11):5994-7. Epub 2012 Aug 13.

Unit of Microbiology, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Italy.

The macrolide-aminoglycoside-streptothricin (MAS) element, an ∼4.2-kb insertion containing erm(B) and aphA3 resistance determinants, distinguishes Streptococcus pneumoniae transposon Tn1545/Tn6003 from Tn6002. Here, it is shown to be an unstable genetic element that, although it lacks recombinase genes, can exploit long, erm(B)-containing direct repeats acting as att sites for spontaneous excision that may result in loss. Consequent to excision, which is RecA independent, Tn1545/Tn6003 changes to Tn6002. In pneumococcal populations harboring Tn1545/Tn6003, the latter appears to coexist with Tn6002.
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http://dx.doi.org/10.1128/AAC.01487-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486530PMC
November 2012

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP.

Proc Natl Acad Sci U S A 2012 Apr 19;109(15):E905-13. Epub 2012 Mar 19.

Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, 9747 AG, Groningen, The Netherlands.

How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type Ser/Thr kinase of S. pneumoniae, StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.
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http://dx.doi.org/10.1073/pnas.1119172109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326482PMC
April 2012

Successful treatment of chronic mucocutaneous candidiasis caused by azole-resistant Candida albicans with posaconazole.

Clin Dev Immunol 2011 1;2011:283239. Epub 2010 Dec 1.

Department of Internal Medicine, Allergy and Clinical Immunology, Azienda Ospedaliero Universitaria, University of Cagliari, SS 554- Bivio Sestu, 09042 Monserrato, Cagliari, Italy.

Refractory or recurrent infections of skin, nails, and the mucous membranes are clinical signs of chronic mucocutaneous candidiasis, frequently associated with immunological defects. Here we describe a 39-years-old female patient, with familial CMC, that presented with an extensive infection caused by an azole-resistant Candida albicans isolate, successfully treated with posaconazole.
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http://dx.doi.org/10.1155/2011/283239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003968PMC
June 2011

Identification of β-lactamases in human and bovine isolates of Staphylococcus aureus strains having borderline resistance to penicillinase-resistant penicillins (PRPs) with proteomic methods.

Vet Microbiol 2011 Jan 18;147(1-2):96-102. Epub 2010 Jun 18.

Department of Human Genetics, University of Debrecen, Debrecen, Hungary.

Methicillin and oxacillin-hydrolyzing enzymes of 6 borderline methicillin-resistant and 1 methicillin-resistant Staphylococcus aureus strains isolated from human clinical samples and 4 borderline methicillin-resistant S. aureus strains isolated from bovine mastitis were investigated. As previous studies suggested the involvement of an additional enzyme besides the penicillinase BlaZ in the determination of borderline resistance, we analyzed the expressed extracellular and membrane-bound β-lactamases with 2-D gel electrophoresis and mass spectrometry. Our analysis showed that the penicillin-hydrolyzing BlaZ alone was responsible for the hydrolysis of both methicillin and oxacillin. All supernatant and membrane fractions contained the same enzyme with slight sequence variations. The size and pI of the proteins were also variable, probably due to spontaneous hydrolysis and/or posttranslational modifications. Interestingly, we found also cytotoxins and other virulence factors in some nitrocefin-hydrolyzing dots, suggesting that those proteins might have a role in the reduction of local antibiotic concentration.
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http://dx.doi.org/10.1016/j.vetmic.2010.06.006DOI Listing
January 2011

Division protein interaction web: identification of a phylogenetically conserved common interactome between Streptococcus pneumoniae and Escherichia coli.

Microbiology (Reading) 2008 Oct;154(Pt 10):3042-3052

Istituto di Biologia e Patologia Molecolare del CNR, Roma, Italy.

The ability of each of the 11 Streptococcus pneumoniae division proteins to interact with itself and with each of the remaining proteins was studied in 66 combinations of protein pairs, using a bacterial two-hybrid system. Interactions (homo- or hetero-dimerizations) were detected between 37 protein pairs, whereas 29 protein pairs did not interact. In some cases, positive interactions of the S. pneumoniae proteins were confirmed by co-immunoprecipitation experiments in Escherichia coli. Comparison between the S. pneumoniae division protein interaction web and that of E. coli, the only micro-organisms for which the whole division interactome has been described systematically, was also performed. At least nine division proteins, ZapA, FtsZ, FtsA, FtsK, FtsQ/DivIB, FtsB/DivIC, FtsL, FtsI and FtsW, are believed to have a conserved function between these bacteria and thus we may say that a significant part of the interactions are conserved. Out of 45 protein pairs tested in both bacteria, 30 showed the same behaviour: 23 interacted while seven did not. In agreement with these results, cross-interactions between S. pneumoniae proteins and the corresponding E. coli orthologues were observed. Taken together, these results suggest a phylogenetically conserved minimal common interactome of the division proteins.
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http://dx.doi.org/10.1099/mic.0.2008/018697-0DOI Listing
October 2008

Roles of pneumococcal DivIB in cell division.

J Bacteriol 2008 Jul 25;190(13):4501-11. Epub 2008 Apr 25.

Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale (Université Joseph Fourier, CNRS UMR 5075, CEA), Grenoble, France.

DivIB, also known as FtsQ in gram-negative organisms, is a division protein that is conserved in most eubacteria. DivIB is localized at the division site and forms a complex with two other division proteins, FtsL and DivIC/FtsB. The precise function of these three bitopic membrane proteins, which are central to the division process, remains unknown. We report here the characterization of a divIB deletion mutant of Streptococcus pneumoniae, which is a coccus that divides with parallel planes. Unlike its homologue FtsQ in Escherichia coli, pneumococcal DivIB is not required for growth in rich medium, but the Delta divIB mutant forms chains of diplococci and a small fraction of enlarged cells with defective septa. However, the deletion mutant does not grow in a chemically defined medium. In the absence of DivIB and protein synthesis, the partner FtsL is rapidly degraded, whereas other division proteins are not affected, pointing to a role of DivIB in stabilizing FtsL. This is further supported by the finding that an additional copy of ftsL restores growth of the Delta divIB mutant in defined medium. Functional mapping of the three distinct alpha, beta, and gamma domains of the extracellular region of DivIB revealed that a complete beta domain is required to fully rescue the deletion mutant. DivIB with a truncated beta domain reverts only the chaining phenotype, indicating that DivIB has distinct roles early and late in the division process. Most importantly, the deletion of divIB increases the susceptibility to beta-lactams, more evidently in a resistant strain, suggesting a function in cell wall synthesis.
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http://dx.doi.org/10.1128/JB.00376-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2446777PMC
July 2008

Streptococcus pneumoniae DivIVA: localization and interactions in a MinCD-free context.

J Bacteriol 2007 Feb 10;189(4):1288-98. Epub 2006 Nov 10.

Dipartimento di Scienze e Tecnologie Biomediche, Sez. Microbiologia Medica, Via Porcell, 4, 09100 Cagliari, Italy.

To clarify the function of DivIVA in Streptococcus pneumoniae, we localized this protein in exponentially growing cells by both immunofluorescence microscopy and immunoelectron microscopy and found that S. pneumoniae DivIVA (DivIVA(SPN)) had a unique localization profile: it was present simultaneously both as a ring at the division septum and as dots at the cell poles. Double-immunofluorescence analysis suggested that DivIVA is recruited to the septum at a later stage than FtsZ and is retained at the poles after cell separation. All the other cell division proteins that we tested were localized in the divIVA null mutant, although the percentage of cells having constricted Z rings was significantly reduced. In agreement with its localization profile and consistent with its coiled-coil nature, DivIVA interacted with itself and with a number of known or putative S. pneumoniae cell division proteins. Finally, a missense divIVA mutant, obtained by allelic replacement, allowed us to correlate, at the molecular level, the specific interactions and some of the facets of the divIVA mutant phenotype. Taken together, the results suggest that although the possibility of a direct role in chromosome segregation cannot be ruled out, DivIVA in S. pneumoniae seems to be primarily involved in the formation and maturation of the cell poles. The localization and the interaction properties of DivIVA(SPN) raise the intriguing possibility that a common, MinCD-independent function evolved differently in the various host backgrounds.
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http://dx.doi.org/10.1128/JB.01168-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797354PMC
February 2007

The fallacies of hope: will we discover new antibiotics to combat pathogenic bacteria in time?

FEMS Microbiol Rev 2006 Nov;30(6):841-52

Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid, Spain.

While newly developed technologies have revolutionized the classical approaches to combating infectious diseases, the difficulties associated with developing novel antimicrobials mean that these technologies have not yet been used to introduce new compounds into the market. The new technologies, including genomics and structural biology, open up exciting possibilities for the discovery of antibiotics. However, a substantial effort to pursue research, and moreover to incorporate the results into the production chain, is required in order to bring new antimicrobials to the final user. In the current scenario of emerging diseases and the rapid spread of antibiotic resistance, an active policy to support these requirements is vital. Otherwise, many valuable programmes may never be fully developed for lack of "interest" and funds (private and public). Will we react in time to avoid potential disaster?
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http://dx.doi.org/10.1111/j.1574-6976.2006.00038.xDOI Listing
November 2006

Analysis of the beta-lactamase plasmid of borderline methicillin-susceptible Staphylococcus aureus: focus on bla complex genes and cadmium resistance determinants cadD and cadX.

Plasmid 2006 Mar 17;55(2):114-27. Epub 2005 Oct 17.

Department of Biomedical Sciences and Technologies, Section of Medical Microbiology, University of Cagliari Medical School, Via Porcell 4, 09100 Cagliari, Italy.

Borderline methicillin-susceptible Staphylococcus aureus strains are a rather homogeneous group, characterized by MICs of penicillinase-resistant penicillins (PRPs) at or just below the susceptibility breakpoint. Other features unique to this group include the presence of a pBW15-like beta-lactamase plasmid, the association with phage complex 94/96, and the production of a PRP-hydrolyzing beta-lactamase activity in addition to the classical penicillinase activity. The four HindIII fragments of pBORa53, a pBW15-like plasmid from the well-studied borderline S. aureus strain a53, were cloned in Escherichia coli, sequenced and analyzed. The plasmid (17,334 bp in size) contains 14 open reading frames (ORFs) and a complete copy of transposon Tn552, which harbors the three genes of the bla complex (blaZ, blaR1, and blaI) necessary for penicillinase production. Among the other 11 ORFs identified, two were homologous to cadmium resistance determinants of Staphylococcus lugdunensis and to the cadD and cadX genes recently detected in S. aureus. Consistent with this, strain a53 was found to be cadmium resistant. From a collection of 30 S. aureus isolates with borderline PRP MIC levels, 27 matched strain a53 in the positive amplification reactions with all of the four primer pairs targeting the cadD-cadX region, the presence of the 17.3-kb plasmid, and the level of cadmium resistance. The well-established S. aureus laboratory strain ATCC 29213 was also found to express cadD-cadX-mediated cadmium resistance. pBORa53 could be re-isolated from transformants obtained by transferring it into a PRP-susceptible recipient. However, while the transformants demonstrated levels of cadmium and penicillin resistance similar to those of strain a53, they remained fully susceptible to PRPs.
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http://dx.doi.org/10.1016/j.plasmid.2005.08.001DOI Listing
March 2006

Altered PBP 2A and its role in the development of penicillin, cefotaxime, and ceftriaxone resistance in a clinical isolate of Streptococcus pneumoniae.

Antimicrob Agents Chemother 2005 May;49(5):2002-7

MRC/NICD/WITS Respiratory and Meningeal Pathogens Research Unit, National Institute for Communicable Diseases, P.O. Box 1038, Johannesburg 2000, South Africa.

We report the unusual involvement of altered PBP 2A in the development of beta-lactam resistance in Streptococcus pneumoniae. This was investigated amid three identical serotype 14 isolates (designated isolates 1, 2, and 3, respectively) of pneumococci cultured successfully from the blood of a human immunodeficiency virus-seropositive child with recurrent pneumonia. The passage of this strain through its human host induced several changes in the bacterium, which is typical of the adaptive and evolving nature of the pneumococcus. An efflux resistance mechanism, which conferred increased ciprofloxacin resistance, was induced in isolates 2 and 3. In addition, faster growth rates and larger capsules were also observed for these isolates, with respect to isolate 1. Notably, compared to isolates 1 and 2, isolate 3 showed a decrease in penicillin, cefotaxime, and ceftriaxone resistance. This change was associated with the replacement of an altered PBP 2A for an unaltered PBP 2A. In all likelihood, these events produced a strain which evolved into a fitter and more virulent type, isolate 3, that resulted in an aggravated pneumococcal infection and ultimately in the patient's death.
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http://dx.doi.org/10.1128/AAC.49.5.2002-2007.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087663PMC
May 2005

Cell division in cocci: localization and properties of the Streptococcus pneumoniae FtsA protein.

Mol Microbiol 2005 Feb;55(3):699-711

Aventis Pharma, 102 Route de Noisy, F-93235 Romainville cedex, France.

We studied the cytological and biochemical properties of the FtsA protein of Streptococcus pneumoniae. FtsA is a widespread bacterial cell division protein that belongs to the actin superfamily. In Escherichia coli and Bacillus subtilis, FtsA localizes to the septal ring after FtsZ, but its exact role in septation is not known. In S. pneumoniae, we found that, during exponential growth, the protein localizes to the nascent septa, at the equatorial zones of the dividing cells, where an average of 2200 FtsA molecules per cell are present. Likewise, FtsZ was found to localize with the same pattern and to be present at an average of 3000 molecules per cell. Consistent with the colocalization, FtsA was found to interact with FtsZ and with itself. Purified FtsA is able to bind several nucleotides, the affinity being highest for adenosine triphosphate (ATP), and lower for other triphosphates and diphosphates. The protein polymerizes in vitro, in a nucleotide-dependent manner, forming long corkscrew-like helixes, composed of 2 + 2 paired protofilaments. No nucleotide hydrolytic activity was detected. Consistent with the absence of an ATPase activity, the polymers are highly stable and not dynamic. These results suggest that the FtsA protein could also polymerize in vivo and the polymers participate in septation.
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http://dx.doi.org/10.1111/j.1365-2958.2004.04432.xDOI Listing
February 2005

Characterization of divIVA and other genes located in the chromosomal region downstream of the dcw cluster in Streptococcus pneumoniae.

J Bacteriol 2003 Oct;185(20):6209-14

Dipartimento di Scienze Chirurgiche Sez. Microbiologia, Università di Cagliari, 09100 Cagliari, Italy.

We analyzed the chromosome region of Streptococcus pneumoniae located downstream of the division and cell wall (dcw) cluster that contains the homolog of the Bacillus subtilis cell division gene divIVA and some genes of unknown function. Inactivation of divIVA in S. pneumoniae resulted in severe growth inhibition and defects in cell shape, nucleoid segregation, and cell division. Inactivation of the ylm genes resulted in some morphological and/or division abnormalities, depending on the inactivated gene. Transcriptional analysis revealed a relationship between these genes and the ftsA and ftsZ cell division genes, also indicating that the connection between the dcw cluster and the divIVA region is more extensive than just chromosomal position and gene organization.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC225046PMC
http://dx.doi.org/10.1128/JB.185.20.6209-6214.2003DOI Listing
October 2003

Phage-display and correlated mutations identify an essential region of subdomain 1C involved in homodimerization of Escherichia coli FtsA.

Proteins 2003 Feb;50(2):192-206

GlaxoSmithKline Medicines Research Center, Via Fleming 4, 37135 Verona, Italy.

FtsA plays an essential role in Escherichia coli cell division and is nearly ubiquitous in eubacteria. Several evidences postulated the ability of FtsA to interact with other septation proteins and with itself. To investigate these binding properties, we screened a phage-display library with FtsA. The isolated peptides defined a degenerate consensus sequence, which in turn displayed a striking similarity with residues 126-133 of FtsA itself. This result suggested that residues 126-133 were involved in homodimerization of FtsA. The hypothesis was supported by the analysis of correlated mutations, which identified a mutual relationship between a group of amino acids encompassing the ATP-binding site and a set of residues immediately downstream to amino acids 126-133. This information was used to assemble a model of a FtsA homodimer, whose accuracy was confirmed by probing multiple alternative docking solutions. Moreover, a prediction of residues responsible for protein-protein interaction validated the proposed model and confirmed once more the importance of residues 126-133 for homodimerization. To functionally characterize this region, we introduced a deletion in ftsA, where residues 126-133 were skipped. This mutant failed to complement conditional lethal alleles of ftsA, demonstrating that amino acids 126-133 play an essential role in E. coli.
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http://dx.doi.org/10.1002/prot.10244DOI Listing
February 2003

Unconventional organization of the division and cell wall gene cluster of Streptococcus pneumoniae.

Microbiology (Reading) 1998 Nov;144 ( Pt 11):3069-3078

Geneva Biomedical Research InstitUte,GlaxoWellcome, 14 Chemin des Aulx, CH-1228 Plans-les-Ouates, Geneva,Switzerland.

The genes responsible for cell wall biosynthesis and cell division (dcw genes) were identified and sequenced in Streptococcus pneumoniae. The genetic organization of the dcw cluster in Streptococcus pneumoniae differed significantly from the clusters of other bacteria reported to date. In particular, the genes corresponding to the 2 min region of the Escherichia coli chromosome were found distributed in three genetically separate regions of the Streptococcus pneumoniae chromosome. The first region contained the expected ftsA and ftsZ cell division genes at one end and pbp2b, ddl and murF at the other end. The murD, murG and divIB genes, always found located upstream of ftsA, were found in a second region separated from the first. A third region contained the yllC, yllD, pbp2x and mraY genes. The chromosomal region downstream of ftsZ was also sequenced and characterized. In Streptococcus pneumoniae this region contains four ORFs, all of unknown function, and an ORF encoding the Bacillus subtilis DivIVA homologue. The gene order and the organization of this region was found to be conserved in Staphylococcus aureus, Streptococcus pyogenes and Bacillus subtilis, raising the possibility that previously unidentified loci may also be involved in division.
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http://dx.doi.org/10.1099/00221287-144-11-3069DOI Listing
November 1998
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