Acinetobacter baumannii phenylacetic acid metabolism influences infection outcome through a direct effect on neutrophil chemotaxis.

Innate cellular immune responses are a critical first-line defense against invading bacterial pathogens. Leukocyte migration from the bloodstream to a site of infection is mediated by chemotactic factors that are often host-derived. More recently, there has been a greater appreciation of the importance of bacterial factors driving neutrophil movement during infection. Here, we describe the development of a zebrafish infection model to study Acinetobacter baumannii pathogenesis. By using isogenic A. baumannii mutants lacking expression of virulence effector proteins, we demonstrated that bacterial drivers of disease severity are conserved between zebrafish and mammals. By using transgenic zebrafish with fluorescent phagocytes, we showed that a mutation of an established A. baumannii global virulence regulator led to marked changes in neutrophil behavior involving rapid neutrophil influx to a localized site of infection, followed by prolonged neutrophil dwelling. This neutrophilic response augmented bacterial clearance and was secondary to an impaired A. baumannii phenylacetic acid catabolism pathway, which led to accumulation of phenylacetate. Purified phenylacetate was confirmed to be a neutrophil chemoattractant. These data identify a previously unknown mechanism of bacterial-guided neutrophil chemotaxis in vivo, providing insight into the role of bacterial metabolism in host innate immune evasion. Furthermore, the work provides a potentially new therapeutic paradigm of targeting a bacterial metabolic pathway to augment host innate immune responses and attenuate disease.

Impact of a Cross-Kingdom Signaling Molecule of Candida albicans on Acinetobacter baumannii Physiology.

Multidrug-resistant (MDR) Acinetobacter baumannii is an opportunistic human pathogen that has become highly problematic in the clinical environment. Novel therapies are desperately required. To assist in identifying new therapeutic targets, the antagonistic interactions between A. baumannii and the most common human fungal pathogen, Candida albicans, were studied. We have observed that the C. albicans quorum-sensing molecule, farnesol, has cross-kingdom interactions, affecting the viability of A. baumannii. To gain an understanding of its mechanism, the transcriptional profile of A. baumannii exposed to farnesol was examined. Farnesol caused dysregulation of a large number of genes involved in cell membrane biogenesis, multidrug efflux pumps (AcrAB-like and AdeIJK-like), and A. baumannii virulence traits such as biofilm formation (csuA, csuB, and ompA) and motility (pilZ and pilH). We also observed a strong induction in genes involved in cell division (minD, minE, ftsK, ftsB, and ftsL). These transcriptional data were supported by functional assays showing that farnesol disrupts A. baumannii cell membrane integrity, alters cell morphology, and impairs virulence characteristics such as biofilm formation and twitching motility. Moreover, we showed that A. baumannii uses efflux pumps as a defense mechanism against this eukaryotic signaling molecule. Owing to its effects on membrane integrity, farnesol was tested to see if it potentiated the activity of the membrane-acting polymyxin antibiotic colistin. When coadministered, farnesol increased sensitivity to colistin for otherwise resistant strains. These data provide mechanistic understanding of the antagonistic interactions between diverse pathogens and may provide important insights into novel therapeutic strategies.

A global virulence regulator in Acinetobacter baumannii and its control of the phenylacetic acid catabolic pathway.

BACKGROUND: Acinetobacter baumannii is one of the most notorious hospital-acquired pathogens, and novel treatment strategies are desperately required. Two-component regulatory systems represent potential therapeutic targets as they mediate microorganism adaptation to changing environments, often control virulence, and are specific to bacteria. Here we describe the first global virulence regulator in A. baumannii. METHODS AND RESULTS: Using transcriptional profiling and functional assays of a deletion mutant in the A. baumannii sensor kinase gene, A1S_0574 (termed as gacS), we show that this sensor kinase regulates key virulence characteristics, including pili synthesis, biofilms, and motility, resulting in virulence attenuation in a mammalian septicemia model. Notably, we also identified that GacS regulates an operon novel to A. baumannii (paa operon), which is responsible for the metabolism of aromatic compounds. Deletion of paaE (A1S_1340) confirmed the role of this operon in A. baumannii virulence. Finally, we identified the cognate response regulator (A1S_0236) for GacS and confirmed their interaction. A1S_0236 was shown to regulate 75% of the GacS transcriptome and the same virulence phenotypes. Overexpression of A1S_0236 restored virulence in the gacS mutant. CONCLUSIONS: Our study characterizes a global virulence regulator, which may provide an alternate therapeutic target, in one of the most troublesome hospital-acquired pathogens.

Carbapenem resistance in Acinetobacter baumannii: laboratory challenges, mechanistic insights and therapeutic strategies.

Unprecedented levels of antimicrobial resistance in bacterial isolates have prompted great concerns globally. In 2012 the WHO released a publication outlining the evolving threat of antimicrobial resistance in order to raise awareness and to stimulate coordinated international efforts. The carbapenem class of antibiotics is largely considered as an antibiotic of last-resort when treating infections. Now carbapenem resistance further limits treatment options. In this article the authors discuss carbapenem resistance in Acinetobacter baumannii, a bacterial isolate often implicated in nosocomial infections. Virulence factors, intrinsic and acquired resistance mechanisms, together with laboratory challenges in the detection and antibiotic susceptibility testing of A. baumannii make this a truly problematic isolate. Therapeutic options are exceedingly limited, relying on polymyxins in combinations with other antibiotics, with few, if any, new active agents in the pipeline.

The success of acinetobacter species; genetic, metabolic and virulence attributes.

An understanding of why certain Acinetobacter species are more successful in causing nosocomial infections, transmission and epidemic spread in healthcare institutions compared with other species is lacking. We used genomic, phenotypic and virulence studies to identify differences between Acinetobacter species. Fourteen strains representing nine species were examined. Genomic analysis of six strains showed that the A. baumannii core genome contains many genes important for diverse metabolism and survival in the host. Most of the A. baumannii core genes were also present in one or more of the less clinically successful species. In contrast, when the accessory genome of an individual A. baumannii strain was compared to a strain of a less successful species (A. calcoaceticus RUH2202), many operons with putative virulence function were found to be present only in the A. baumannii strain, including the csu operon, the acinetobactin chromosomal cluster, and bacterial defence mechanisms. Phenotype microarray analysis showed that compared to A. calcoaceticus (RUH2202), A. baumannii ATCC 19606(T) was able to utilise nitrogen sources more effectively and was more tolerant to pH, osmotic and antimicrobial stress. Virulence differences were also observed, with A. baumannii ATCC 19606(T), A. pittii SH024, and A. nosocomialis RUH2624 persisting and forming larger biofilms on human skin than A. calcoaceticus. A. baumannii ATCC 19606(T) and A. pittii SH024 were also able to survive in a murine thigh infection model, whereas the other two species were eradicated. The current study provides important insights into the elucidation of differences in clinical relevance among Acinetobacter species.

Insights into Acinetobacter baumannii pathogenicity.

Acinetobacter spp. have justifiably received significant attention from the public, scientific, and medical communities. Over recent years, Acinetobacter, particularly Acinetobacter baumannii, has become a "red-alert" human pathogen, primarily because of its exceptional ability to develop resistance to all currently available antibiotics. This characteristic is compounded by its unique abilities to survive in a diverse range of environments, including those within healthcare institutions, leading to problematic outbreaks. Historically, the virulence of the organism has been questioned, but recent clinical reports suggest that Acinetobacter can cause serious, life-threatening infections. Furthermore, its metabolic adaptability gives it a selective advantage in harsh hospital environments. This review focuses on current understanding of A. baumannii pathogenesis and the model systems used to study this interesting organism.

Inactivation of clpB in the pathogen Leptospira interrogans reduces virulence and resistance to stress conditions.

Leptospira interrogans is the causative agent of leptospirosis, which is an emerging zoonotic disease. Resistance to stress conditions is largely uncharacterized for this bacterium. We therefore decided to analyze a clpB mutant that we obtained by random transposon mutagenesis. The mutant did not produce any of the two isoforms of ClpB. The clpB mutant exhibited growth defects at 30° and 37°C and in poor nutrient medium and showed increased susceptibility to oxidative stress, whereas the genetically complemented strain was restored in ClpB expression and in vitro wild-type growth. We also showed that the clpB mutant was attenuated in virulence in an animal model of acute leptospirosis. Our findings demonstrate that ClpB is involved in the general stress response. The chaperone is also necessary, either directly or indirectly, for the virulence of the pathogen L. interrogans.

Development of transcriptional fusions to assess Leptospira interrogans promoter activity.

BACKGROUND: Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field. METHODOLOGY AND PRINCIPAL FINDINGS: A series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP) were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA) and Sphingomyelinase 2 (sph2) promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain. CONCLUSIONS: The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa.

Development of Transcriptional Fusions to Assess Leptospira interrogans Promoter Activity

Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic toolsfor Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction ofpathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field. A series of promoter-probe vectors carrying a reporter gene encoding greenfluorescent protein (GFP) were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA) and Sphingomielynase 2 (sph2) promoters from L. interrogansand the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the noninduced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain. The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflectedtranscriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa.

Characterization of the immunogenic and antigenic potential of putative lipoproteins from Leptospira interrogans.

The search for a vaccine capable of conferring heterologous protection, through the identification of conserved and cross-protective antigens, remains an ongoing priority in leptospirosis research. In the present study, an in silico analysis was used to identify potentially protective lipoproteins from Leptospira interrogans serovar Copenhageni. Eight putative lipoproteins were selected (LIC10009, LIC10054, LIC10091, LIC11058, LIC11567, LIC13059, LIC13305, and LIC20172), cloned and expressed in Escherichia coli and purified by affinity chromatography. The recombinant proteins were used to inoculate mice and the subsequent humoral immune response was evaluated by ELISA. Seven of the potential lipoproteins induced a significant IgG response. Furthermore, all of the recombinant proteins were recognized by antibodies present in the sera of severe leptospirosis patients. These putative lipoproteins exhibited potential for further evaluation as prospective vaccine candidates.

Bioinformatics describes novel Loci for high resolution discrimination of leptospira isolates.

BACKGROUND: Leptospirosis is one of the most widespread zoonoses in the world and with over 260 pathogenic serovars there is an urgent need for a molecular system of classification. The development of multilocus sequence typing (MLST) schemes for Leptospira spp. is addressing this issue. The aim of this study was to identify loci with potential to enhance Leptospira strain discrimination by sequencing-based methods. METHODOLOGY AND PRINCIPAL FINDINGS: We used bioinformatics to evaluate pre-existing loci with the potential to increase the discrimination of outbreak strains. Previously deposited sequence data were evaluated by phylogenetic analyses using either single or concatenated sequences. We identified and evaluated the applicability of the ligB, secY, rpoB and lipL41 loci, individually and in combination, to discriminate between 38 pathogenic Leptospira strains and to cluster them according to the species they belonged to. Pairwise identity among the loci ranged from 82.0-92.0%, while interspecies identity was 97.7-98.5%. Using the ligB-secY-rpoB-lipL41 superlocus it was possible to discriminate 34/38 strains, which belong to six pathogenic Leptospira species. In addition, the sequences were concatenated with the superloci from 16 sequence types from a previous MLST scheme employed to study the association of a leptospiral clone with an outbreak of human leptospirosis in Thailand. Their use enhanced the discriminative power of the existing scheme. The lipL41 and rpoB loci raised the resolution from 81.0-100%, but the enhanced scheme still remains limited to the L. interrogans and L. kirschneri species. CONCLUSIONS: As the first aim of our study, the ligB-secY-rpoB-lipL41 superlocus demonstrated a satisfactory level of discrimination among the strains evaluated. Second, the inclusion of the rpoB and lipL41 loci to a MLST scheme provided high resolution for discrimination of strains within L. interrogans and L. kirschneri and might be useful in future epidemiological studies.

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG.

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18 kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.

Expanding the genetic toolbox for Leptospira species by generation of fluorescent bacteria.

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-ß-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.

Preliminary characterization of Mus musculus-derived pathogenic strains of Leptospira borgpetersenii serogroup Ballum in a hamster model.

Human and animal leptospirosis caused by Leptospira spp. belonging to serogroup Ballum has increased worldwide in the past decade. We report the isolation and serologic and molecular characterization of four L. borgpetersenii serogroup Ballum isolates obtained from Mus musculus, and preliminary virulence studies. These isolates are useful for diagnosis of leptospirosis and for epidemiologic studies of its virulence and pathogenic mechanisms.

LepBank: a Leptospira sequence repository and a portal for phylogenetic studies.

Leptospirosis is a neglected infectious disease that constitutes a threat to both humans and animals. Comprehension about the epidemiological behavior and population dynamics of Leptospira may be helpful for the development of control measures. Thus, an effort was made to organize leptospiral sequences in a new and specific database. In addition, online bioinformatics tools were clustered in a web portal to facilitate sequences manipulation by scientists. LepBank (http://.lepbank.ufpel.edu.br) is a Leptospira sequences repository and a suite for systematics, which brings simplicity to leptospirosis research, integrating sophisticated online programs to a sequence database. We intend the database to be useful for the leptospirosis scientific community, providing standardized and high quality information and facilitating research into key aspects of the Leptospira taxonomy and phylogeny.

Monitoring Leptospira strain collections: the need for quality control.

The purpose of this study was to perform a 16S sequence-based quality control of two Leptospira strain collections. 16S rRNA gene sequencing was used to verify two Leptospira reference collections provided by the World Health Organization and maintained at a reference laboratory for leptospirosis in Brazil. Among the 89 serovars evaluated, four conflicting strains were identified in one of the collections. Although 16S rRNA gene sequencing cannot identify Leptospira beyond the species level, it is suitable for the identification of contamination and quality control of leptospiral reference collections. This study highlights the importance of the availability of high-quality 16S rRNA sequences in public databases. In addition, it emphasizes the need for periodical verifications and quality control of Leptospira reference collections.

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.

A century of Leptospira strain typing.

Leptospirosis is a zoonotic disease with high mortality and morbidity rates in humans and animals throughout the world. Since the discovery of Leptospira, the causal agent of leptospirosis, a century ago, this spirochete has been isolated from the environment and a wide spectrum of animals and classified into serogroups and serovars as a function of antigenic determinants. Modern technology has greatly improved laboratory procedures, particularly those for the detection, identification and typing of epidemiologic strains. In this review, we describe "classical" serotyping methods, followed by a description of genotyping and post-genomic typing methods.

Distribution of the leptospiral immunoglobulin-like (lig) genes in pathogenic Leptospira species and application of ligB to typing leptospiral isolates.

The family of leptospiral immunoglobulin-like (lig) genes comprises ligA, ligB and ligC. This study used PCR to demonstrate the presence of lig genes among serovars from a collection of leptospiral strains and clinical isolates. Whilst ligA and ligC appeared to be present in a limited number of pathogenic serovars, the ligB gene was distributed ubiquitously among all pathogenic strains. None of the lig genes were detected among intermediate or saprophytic Leptospira species. It was also shown that, similar to the previously characterized secY gene, a short specific PCR fragment of ligB could be used to correctly identify pathogenic Leptospira species. These findings demonstrate that ligB is widely present among pathogenic strains and may be useful for their reliable identification and classification.