Sensitizing Staphylococcus aureus to antibacterial agents by decoding and blocking the lipid flippase MprF.

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. Multiple peptide resistance factor (MprF) is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to antimicrobial agents and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding of bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin, and it impaired MRSA survival in human phagocytes. Thus, MprF inhibitors are recommended for new antivirulence approaches against MRSA and other bacterial pathogens.

Sequence of the R1 plasmid and comparison to F and R100.

The R1 antibiotic resistance plasmid, originally discovered in a clinical Salmonella isolate in London, 1963, has served for decades as a key model for understanding conjugative plasmids. Despite its scientific importance, a complete sequence of this plasmid has never been reported. We present the complete genome sequence of R1 along with a brief review of the current knowledge concerning its various genetic systems and a comparison to the F and R100 plasmids. R1 is 97,566 nucleotides long and contains 120 genes. The plasmid consists of a backbone largely similar to that of F and R100, a Tn21-like transposon that is nearly identical to that of R100, and a unique 9-kb sequence that bears some resemblance to sequences found in certain Klebsiella oxytoca strains. These three regions of R1 are separated by copies of the insertion sequence IS1. Overall, the structure of R1 and comparison to F and R100 suggest a fairly stable shared conjugative plasmid backbone into which a variety of mobile elements have inserted to form an "accessory" genome, containing multiple antibiotic resistance genes, transposons, remnants of phage genes, and genes whose functions remain unknown.

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α.

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

Coupling between the basic replicon and the Kis-Kid maintenance system of plasmid R1: modulation by Kis antitoxin levels and involvement in control of plasmid replication.

kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

Transfer of the amino-terminal nuclear envelope targeting domain of human MX2 converts MX1 into an HIV-1 resistance factor.

UNLABELLED: The myxovirus resistance 2 (MX2) protein of humans has been identified recently as an interferon (IFN)-inducible inhibitor of human immunodeficiency virus type 1 (HIV-1) that acts at a late postentry step of infection to prevent the nuclear accumulation of viral cDNA (C. Goujon et al., Nature 502:559-562, 2013, http://dx.doi.org/10.1038/nature12542; M. Kane et al., Nature 502:563-566, 2013, http://dx.doi.org/10.1038/nature12653; Z. Liu et al., Cell Host Microbe 14:398-410, 2013, http://dx.doi.org/10.1016/j.chom.2013.08.015). In contrast, the closely related human MX1 protein, which suppresses infection by a range of RNA and DNA viruses (such as influenza A virus [FluAV]), is ineffective against HIV-1. Using a panel of engineered chimeric MX1/2 proteins, we demonstrate that the amino-terminal 91-amino-acid domain of MX2 confers full anti-HIV-1 function when transferred to the amino terminus of MX1, and that this fusion protein retains full anti-FluAV activity. Confocal microscopy experiments further show that this MX1/2 fusion, similar to MX2 but not MX1, can localize to the nuclear envelope (NE), linking HIV-1 inhibition with MX accumulation at the NE. MX proteins are dynamin-like GTPases, and while MX1 antiviral function requires GTPase activity, neither MX2 nor MX1/2 chimeras require this attribute to inhibit HIV-1. This key discrepancy between the characteristics of MX1- and MX2-mediated viral resistance, together with previous observations showing that the L4 loop of the stalk domain of MX1 is a critical determinant of viral substrate specificity, presumably reflect fundamental differences in the mechanisms of antiviral suppression. Accordingly, we propose that further comparative studies of MX proteins will help illuminate the molecular basis and subcellular localization requirements for implementing the noted diversity of virus inhibition by MX proteins. IMPORTANCE: Interferon (IFN) elicits an antiviral state in cells through the induction of hundreds of IFN-stimulated genes (ISGs). The human MX2 protein has been identified as a key effector in the suppression of HIV-1 infection by IFN. Here, we describe a molecular genetic approach, using a collection of chimeric MX proteins, to identify protein domains of MX2 that specify HIV-1 inhibition. The amino-terminal 91-amino-acid domain of human MX2 confers HIV-1 suppressor capabilities upon human and mouse MX proteins and also promotes protein accumulation at the nuclear envelope. Therefore, these studies correlate the cellular location of MX proteins with anti-HIV-1 function and help establish a framework for future mechanistic analyses of MX-mediated virus control.

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells.

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum ß-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.

Spread of CTX-M-15 positive Providencia spp. causing urinary tract infections at the University Hospital Split in Croatia.

AIM: During 2010-2011, six Providencia spp. (five Providencia stuartii and one Providencia rettgeri) urine isolates with unusual resistance phenotype were collected from various hospital units at the University Hospital Split in Croatia. The aim of the study was to analyze the mechanisms of resistance to expanded-spectrum cephalosporins. METHODS: The antimicrobial susceptibility to a wide range of antibiotics was determined by broth microdilution method according to CLSI guidelines. A double-disk-synergy test (DDST) was performed to detect ESBLs. The transferability of cefotaxime resistance was determined by conjugation. The presence of genes encoding ESBLs was determined by PCR while genotyping of the isolates was performed by PFGE. RESULTS: All strains were positive for ESBL production by DDST. They were uniformly resistant to amoxycillin alone and combined with clavulanate, cefazoline, cefuroxime, ceftazidime, cefotaxime, ceftriaxone, gentamicin and ciprofloxacin. P. stuartii strains transferred cefotaxime resistance to E. coli recipient strain with frequency ranging from 10-5 to 5x10-4. Five P. stuartii strains were positive for TEM and CTX-M ß-lactamases while P. rettgeri was positive only for TEM ß-lactamases. Five CTX-M producing isolates were shown to be clonally related. CONCLUSIONS: Continuous surveillance in tracking CTX-M-15- producing P. stuartii in the hospitals is necessary to prevent their spread to other hospitals and community. Global spread of ESBL positive Providencia spp all over the world is of great clinical concern.

Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication.

The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42°C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.

Kis antitoxin couples plasmid R1 replication and parD (kis,kid) maintenance modules.

The coupling between the replication and parD (kis, kid) maintenance modules of R1 has been revisited here by the isolation of a significant collection of conditional replication mutants in the pKN1562 mini-R1 plasmid, and in its derivative, pJLV01, specifically affected in the RNase activity of the Kid toxin. This new analysis aims to identify key factors in this coupling. For this purpose we have quantified and characterized the restriction introduced by parD to isolate conditional replication mutants of this plasmid, a signature of the modular coupling. This restriction depends on the RNase activity of the Kid toxin and it is relieved by either over-expression of the Kis antitoxin or by preventing its degradation by Lon and ClpAP proteases. Based on these data and on the correlation between copy numbers and parD transcriptional levels obtained in the different mutants, it is proposed that a reduction of Kis antitoxin levels in response to inefficient plasmid replication is the key factor for coupling plasmid replication and parD modules.

General requirements for protein secretion by the F-like conjugation system R1.

Bacterial conjugation disseminates genes among bacteria via a process requiring direct cell contact. The cell envelope spanning secretion apparatus involved belongs to the type IV family of bacterial secretion systems, which transport protein as well as nucleoprotein substrates. This study aims to understand mechanisms leading to the initiation of type IV secretion using conjugative plasmid paradigm R1. We analyze the general requirements for plasmid encoded conjugation proteins and DNA sequence within the origin of transfer (oriT) for protein secretion activity using a Cre recombinase reporter system. We find that similar to conjugative plasmid DNA strand transfer, activation of the R1 system for protein secretion depends on binding interactions between the multimeric, ATP-binding coupling protein and the R1 relaxosome including an intact oriT. Evidence for DNA independent protein secretion was not found.

Extended-spectrum ß-lactamase-producing enterobacteriaceae causing nosocomial infections in Mexico. A retrospective and multicenter study.

BACKGROUND AND AIMS: Extended-spectrum ß-lactamase (ESBLs) production is still the most frequent mechanism of resistance to cephalosporins in gram-negative bacteria. The aim of the study was to identify the types of ESBL-producing Enterobacteriaceae clinical isolates causing nosocomial infections in Mexico. METHODS: ESBL production was performed using a disk diffusion method. The MIC for several antibiotics was performed by agar dilution on Mueller-Hinton. PFGE typing was carried out on all enterobacteria assayed. The ß-lactamase pattern was obtained by IEF and bioassay. Genes of ß-lactamases were amplified by PCR with specific primers and products were sequenced and analyzed using informatics programs. Plasmid isolation and conjugation experiments were carried out using standard methodologies. RESULTS: There were 134 isolates of Enterobacteriaceae included from a retrospective and multicenter study that included eight Mexican hospitals from 1999 to 2005. The most prevalent species were K. pneumoniae (56%), Enterobacter cloacae (29%), and Escherichia coli (15%). Molecular analysis identified the underlying endemic and polyclonal spread of enterobacterials in each hospital. The most frequent ESBLs identified were SHV-type (84%), TLA-1 (11%), and CTX-M-15 (5%). Successful matings were detected in 68.4% (71/104) isolates. CONCLUSIONS: ESBL-producer K. pneumoniae remains the most frequent bacterial species obtained in nosocomial infections. The SHV-type and TLA-1 ESBLs were disseminated in most hospitals analyzed and CTX-M-15 was emerging in one of the studied hospitals. This work highlights the proper use of antibiotics to avoid the selection of these types of multiresistant bacteria.

Pentapeptide-repeat proteins that act as topoisomerase poison resistance factors have a common dimer interface.

The protein AlbG is a self-resistance factor against albicidin, a nonribosomally encoded hybrid polyketide-peptide with antibiotic and phytotoxic properties produced by Xanthomonas albilineans. Primary-sequence analysis indicates that AlbG is a member of the pentapeptide-repeat family of proteins (PRP). The structure of AlbG from X. albilineans was determined at 2.0 Šresolution by SAD phasing using data collected from a single trimethyllead acetate derivative on a home source. AlbG folds into a right-handed quadrilateral ß-helix composed of approximately eight semi-regular coils. The regularity of the ß-helix is blemished by a large loop/deviation in the ß-helix between coils 4 and 5. The C-terminus of the ß-helix is capped by a dimerization module, yielding a dimer with a 110 Šsemi-collinear ß-helical axis. This method of dimer formation appears to be common to all PRP proteins that confer resistance to topoisomerase poisons and contrasts with most PRP proteins, which are typically monomeric.

Functional analysis of the finO distal region of plasmid R1.

The intergenic region linking conjugative transfer and replication copy control modules of IncF plasmids shows conservation of gene homology and organization. Genes distal to finO are coordinately expressed with the upstream transfer operon encoding the majority of conjugation genes in related plasmids. Here we investigate potential functions for these genes in copy number control and in processes related to conjugation: gene transfer, pilus specific phage infection and plasmid-promoted biofilm formation by an Escherichia coli host. We find that insertional inactivation of genes in the finO distal region reduced transcriptional read through into the downstream copB gene of plasmid R1. The mutant plasmid derivatives exhibited a reduced copy number compared to the wild type. Moreover all insertion mutant derivatives of plasmid R1-16 with aberrantly low copy numbers conferred poor biofilm forming ability to their hosts. The general mutagenesis thus identified plasmid stability genes as the only plasmid functions besides conjugation genes linked to plasmid-promoted biofilm production under these laboratory conditions. Our findings imply that a novel component of cis- or trans-regulation on the transcriptional level is important to normal R1 plasmid copy number regulation.

The transfer operon of plasmid R1 extends beyond finO into the downstream replication genes.

finO is the final gene in the 35.4 kb transfer operon of IncFI plasmid F that is known to be involved in self-conjugative transfer. The genetic region distal to finO separates the conjugation and replication control modules of IncFII plasmid R100 and carries uncharacterized genes not found in plasmid F. However, comparison of the R100 gene organization with database entries of F-like plasmids suggests its broad conservation. We determined the DNA sequence of this region of IncFII plasmid R1 and studied its transcriptional organization. We find that transcription occurs through the entire gene region spanning from finO to the 5' regulatory region of copB. The region lacks independent promoter activity and transcription is co-regulated with the adjacent transfer operon. We show that this extended transcriptional organization beyond finO is shared by plasmid R100. These findings support the hypothesis that gene products coexpressed from the finO-distal region in F-like plasmids are advantageous under some conditions when conjugative DNA is exchanged.

Gene and cell survival: lessons from prokaryotic plasmid R1.

Plasmids are units of extrachromosomal genetic inheritance found in all kingdoms of life. They replicate autonomously and undergo stable propagation in their hosts. Despite their small size, plasmid replication and gene expression constitute a metabolic burden that compromises their stable maintenance in host cells. This pressure has driven the evolution of strategies to increase plasmid stability--a process accelerated by the ability of plasmids to transfer horizontally between cells and to exchange genetic material with their host and other resident episomal DNAs. These abilities drive the adaptability and diversity of plasmids and their host cells. Indeed, survival functions found in plasmids have chromosomal homologues that have an essential role in cellular responses to stress. An analysis of these functions in the prokaryotic plasmid R1, and of their intricate interrelationships, reveals remarkable overall similarities with other gene- and cell-survival strategies found within and beyond the prokaryotic world.

Reconstitution of DNA segregation driven by assembly of a prokaryotic actin homolog.

Multiple unrelated polymer systems have evolved to partition DNA molecules between daughter cells at division. To better understand polymer-driven DNA segregation, we reconstituted the three-component segregation system of the R1 plasmid from purified components. We found that the ParR/parC complex can construct a simple bipolar spindle by binding the ends of ParM filaments, inhibiting dynamic instability, and acting as a ratchet permitting incorporation of new monomers and riding on the elongating filament ends. Under steady-state conditions, the dynamic instability of unattached ParM filaments provides the energy required to drive DNA segregation.

Structure and function of bacterial kid-kis and related toxin-antitoxin systems.

Toxin-antitoxin systems were discovered as plasmid auxiliary maintenance cassettes. In recent years, an increasing amount of structural and functional information has become available about the proteins involved, allowing the understanding of bacterial cell growth inhibition by the toxins on a molecular level. A well-studied TA system is formed by the proteins Kid and Kis, encoded by the parD operon of the Escherichia coli plasmid R1. The toxicity of Kid has been related to its endoribonuclease activity, which is counteracted by binding of the antitoxin Kis at the proposed active site. In this review, the structural studies on the Kid-Kis system are compared to those of three related toxin-antitoxin systems: MazF-MazE, CcdB-CcdA and RelE-RelB.

Bacterial elicitation and evasion of plant innate immunity.

Recent research on plant responses to bacterial attack has identified extracellular and intracellular host receptors that recognize conserved pathogen-associated molecular patterns and more specialized virulence proteins, respectively. These findings have shed light on our understanding of the molecular mechanisms by which bacteria elicit host defences and how pathogens have evolved to evade or suppress these defences.