Antibiotic persistence of intracellular Brucella abortus

Human brucellosis caused by the facultative intracellular pathogen Brucella spp. is an endemic bacterial zoonosis manifesting as acute or chronic infections with high morbidity. Treatment typically involves a combination therapy of two antibiotics for several weeks to months, but despite this harsh treatment relapses occur at a rate of 5-15%. Although poor compliance and reinfection may account for a fraction of the observed relapse cases, it is apparent that the properties of the infectious agent itself may play a decisive role in this phenomenon.; We used B. abortus carrying a dual reporter in a macrophage infection model to gain a better understanding of the efficacy of recommended therapies in cellulo. For this we used automated fluorescent microscopy as a prime read-out and developed specific CellProfiler pipelines to score infected macrophages at the population and the single cell level. Combining microscopy of constitutive and induced reporters with classical CFU determination, we quantified the protective nature of the Brucella intracellular lifestyle to various antibiotics and the ability of B. abortus to persist in cellulo despite harsh antibiotic treatments.; We demonstrate that treatment of infected macrophages with antibiotics at recommended concentrations fails to fully prevent growth and persistence of B. abortus in cellulo, which may be explained by a protective nature of the intracellular niche(s). Moreover, we show the presence of bona fide intracellular persisters upon antibiotic treatment, which are metabolically active and retain the full infectious potential, therefore constituting a plausible reservoir for reinfection and relapse. In conclusion, our results highlight the need to extend the spectrum of models to test new antimicrobial therapies for brucellosis to better reflect the in vivo infection environment, and to develop therapeutic approaches targeting the persister subpopulation

Gene Transfer Agent Promotes Evolvability within the Fittest Subpopulation of a Bacterial Pathogen

The Bartonella gene transfer agent (BaGTA) is an archetypical example for domestication of a phage-derived element to permit high-frequency genetic exchange in bacterial populations. Here we used multiplexed transposon sequencing (TnSeq) and single-cell reporters to globally define the core components and transfer dynamics of BaGTA. Our systems-level analysis has identified inner- and outer-circle components of the BaGTA system, including 55 regulatory components, as well as an additional 74 and 107 components mediating donor transfer and recipient uptake functions. We show that the stringent response signal guanosine-tetraphosphate (ppGpp) restricts BaGTA induction to a subset of fast-growing cells, whereas BaGTA particle uptake depends on a functional Tol-Pal trans-envelope complex that mediates outer-membrane invagination upon cell division. Our findings suggest that Bartonella evolved an efficient strategy to promote genetic exchange within the fittest subpopulation while disfavoring exchange of deleterious genetic information, thereby facilitating genome integrity and rapid host adaptation

A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

Brucella, the agent causing brucellosis, is a major zoonotic pathogen with worldwide distribution. Brucella resides and replicates inside infected host cells in membrane-bound compartments called Brucella- containing vacuoles (BCVs). Following uptake, Brucella resides in endosomal BCVs (eBCVs) that gradually mature from early to late endosomal features. Through a poorly understood process that is key to the intracellular lifestyle of Brucella, the eBCV escapes fusion with lysosomes by transitioning to the replicative BCV (rBCV), a replicative niche directly connected to the endoplasmic reticulum (ER). Despite the notion that this complex intracellular lifestyle must depend on a multitude of host factors, a holistic view on which of these components control Brucella cell entry, trafficking, and replication is still missing. Here we used a systematic cell-based small interfering RNA (siRNA) knockdown screen in HeLa cells infected with Brucella abortus and identified 425 components of the human infectome for Brucella infection. These include multiple components of pathways involved in central processes such as the cell cycle, actin cytoskeleton dynamics, or vesicular trafficking. Using assays for pathogen entry, knockdown complementation, and colocalization at single-cell resolution, we identified the requirement of the VPS retromer for Brucella to escape the lysosomal degradative pathway and to establish its intracellular replicative niche. We thus validated the VPS retromer as a novel host factor critical for Brucella intracellular trafficking. Further, our genomewide data shed light on the interplay between central host processes and the biogenesis of the Brucella replicative niche.; IMPORTANCE; With >300,000 new cases of human brucellosis annually, Brucella is regarded as one of the most important zoonotic bacterial pathogens worldwide. The agent causing brucellosis resides inside host cells within vacuoles termed Brucella- containing vacuoles (BCVs). Although a few host components required to escape the degradative lysosomal pathway and to establish the ER-derived replicative BCV (rBCV) have already been identified, the global understanding of this highly coordinated process is still partial, and many factors remain unknown. To gain deeper insight into these fundamental questions, we performed a genomewide RNA interference (RNAi) screen aiming at discovering novel host factors involved in the Brucella intracellular cycle. We identified 425 host proteins that contribute to Brucella cellular entry, intracellular trafficking, and replication. Together, this study sheds light on previously unknown host pathways required for the Brucella infection cycle and highlights the VPS retromer components as critical factors for the establishment of the Brucella intracellular replicative niche

An integrative strategy to identify the entire protein coding potential of prokaryotic genomes by proteogenomics

Accurate annotation of all protein-coding sequences (CDSs) is an essential prerequisite to fully exploit the rapidly growing repertoire of completely sequenced prokaryotic genomes. However, large discrepancies among the number of CDSs annotated by different resources, missed functional short open reading frames (sORFs), and overprediction of spurious ORFs represent serious limitations. Our strategy toward accurate and complete genome annotation consolidates CDSs from multiple reference annotation resources, ab initio gene prediction algorithms and in silico ORFs (a modified six-frame translation considering alternative start codons) in an integrated proteogenomics database (iPtgxDB) that covers the entire protein-coding potential of a prokaryotic genome. By extending the PeptideClassifier concept of unambiguous peptides for prokaryotes, close to 95% of the identifiable peptides imply one distinct protein, largely simplifying downstream analysis. Searching a comprehensive Bartonella henselae proteomics data set against such an iPtgxDB allowed us to unambiguously identify novel ORFs uniquely predicted by each resource, including lipoproteins, differentially expressed and membrane-localized proteins, novel start sites and wrongly annotated pseudogenes. Most novelties were confirmed by targeted, parallel reaction monitoring mass spectrometry, including unique ORFs and single amino acid variations (SAAVs) identified in a re-sequenced laboratory strain that are not present in its reference genome. We demonstrate the general applicability of our strategy for genomes with varying GC content and distinct taxonomic origin. We release iPtgxDBs for B. henselae, Bradyrhizobium diazoefficiens and Escherichia coli and the software to generate both proteogenomics search databases and integrated annotation files that can be viewed in a genome browser for any prokaryote

Uncovering the transcriptional control of "Bartonella henselae" host adaptation factors

A recurrent theme in bacterial pathogenicity is the understanding of the regulatory events necessary for a given pathogen to progress through its infection cycle while resisting the host defense mechanisms. This progression typically requires the coordinated expression of defined sub-portions of the virulence repertoire at the same time as others need to be tightly repressed or degraded. This so-called adaptive response is ultimately linked to the ability of the pathogen to sense its direct environment and to transduce this information into the appropriate cellular response. Bacteria have evolved numerous dedicated mechanisms for perception and signal transduction that are characterized by a wide range of signal specificity. Not surprisingly, most of these systems have been adopted by pathogenic bacteria to modulate the expression of their virulence factors. In this work, we present the results of our investigations on the mounting and the regulation of the adaptive response of the zoonotic bacterial pathogen Bartonella henselae to its eukaryotic host. The VirB/D4 type IV secretion system (T4SS) is an essential machinery for the host adaptation of this stealthy pathogen. Using the regulation of this pathogenicity factor as a red thread we uncovered two critical signal transductions pathways that enable B. henselae to coordinate the expression of its virulence factors through its infection cycle. In the research article I, we describe the adaptive response of B. henselae during host cell infection and reveal the central role of the BatR/BatS two component system (TCS) for the coordination of this response. We demonstrate that this TCS is activated at the physiological pH of blood (pH7.4) and is required for the up-regulation of a critical cluster of genes that includes the genes encoding the VirB/D4 T4SS and its cognate secreted effectors (Beps). In the research article II, we present the near complete expressed proteome of B. henselae under conditions that mimic host-interaction, using a combination of saturated transcriptome profiling by RNA-seq and directed shotgun proteomics. Of particular interest, the complete membrane proteome coverage achieved reveals the dramatic re-organization taking place in this compartment during the infection process, with the differential regulation of a large panel of autotransporters, adhesins and hemin binding proteins as well as all components of the VirB/D4 T4SS. In the research article III, we describe how a dual regulatory input controls the expression of the VirB/D4 T4SS and its secreted effector proteins. We demonstrate that additionally to the BatR/BatS TCS, the expression of this host adaptation factors requires the alternative sigma factor RpoH1, which is itself controlled by the stringent response (SR) components SpoT and DksA. In contrast to the VirB/D4 T4SS, which is needed at the early stage of mammalian host colonization and require the SR components for its full expression, we show that SpoT and DksA negatively regulate the Trw T4SS, which is required for erythrocyte invasion at a later stage of the host infection. In the research article IV, we demonstrate the possible use of B. henselae to deliver DNA into human cells through its VirB/D4 T4SS and to generate stable transgenic cell lines. We propose that due to its ancestral abilities as conjugation system, this specialized transkingdom secretion system has potential for the development of new in vivo gene therapy approaches in humans. Together, these results constitute the first comprehensive analysis of B. henselae pathogenicity factors during host cell infection. Besides the elucidation of very specific regulatory aspects for the expression of the VirB/D4 T4SS and its secreted substrates, this work allows us to propose a general model for B. henselae host adaptation strategy throughout its infection cycle. In our model, the BatR/BatS pH-dependent signaling is used to distinguish between the arthropod and the mammalian host environment whereas the SR signaling allows the modulation of the bacterial response between the early and the late colonization stages of the mammalian host

Bartonella gene transfer agent: Evolution, function, and proposed role in host adaptation

The processes underlying host-adaptation by bacterial pathogens remain a fundamental question with relevant clinical, ecological and evolutionary implications. Zoonotic pathogens of the genus Bartonella constitute an exceptional model to study these aspects. Bartonellae have undergone a spectacular diversification into multiple species resulting from adaptive radiation. Specific adaptations of a complex facultative intracellular lifestyle have enabled the colonization of distinct mammalian reservoir hosts. This remarkable host adaptability has a multifactorial basis and is thought to be driven by horizontal gene transfer (HGT) and recombination among a limited genus-specific pan-genome. Recent functional and evolutionary studies revealed that the conserved Bartonella gene transfer agent (BaGTA) mediates highly efficient HGT and could thus drive this evolution. Here we review the recent progress made towards understanding BaGTA evolution, function, and its role in the evolution and pathogenesis of Bartonella spp.. We notably discuss how BaGTA could have contributed to genome diversification through recombination of beneficial traits that underlie host adaptability. We further address how BaGTA may counter the accumulation of deleterious mutations in clonal populations (Muller's Ratchet), that are expected to occur through the recurrent transmission bottlenecks during the complex infection cycle of these pathogens in their mammalian reservoir hosts and arthropod vectors

Systems-level interference strategies to decipher host factors involved in bacterial pathogen interaction: from RNAi to CRISPRi

Bacterial pathogen-host cell interactions involve an intricate interplay of multiple components from both partners. Systems level surveys have been used widely to profile host requirements for pathogen infection. Functional genomics, and more specifically genome-wide perturbation screens, constitute attractive methodologies to assess such host infectomes. Although these strategies have successfully identified numerous critical host factors, they may have failed in generating the high-quality data required for systems level analysis. This is the case for most RNA interference (RNAi) setups with their high propensity to off-target effects (OTE). However, recent efforts to circumvent OTE in RNAi-based experiments as well as the emergence of alternative strategies will likely soon allow significant breakthrough in the systems level understanding of bacterial pathogen-host interactions

TA module toxins as <i>bona fide</i> substrates for conjugative type IV secretion.

<p>(A) The position of known and proposed T4SS substrates as well as proteins with a Bep-like β-hairpin are highlighted in the phylogeny of FicT toxins (adapted from our previous work [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.ref021" target="_blank">21</a>]; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.s003" target="_blank">S3A–S3C Fig</a>). In short, the tree represents a maximum likelihood phylogeny that had been constructed from an alignment of the toxins’ FIC domains. An additional phylogeny supporting the repeated, independent recruitment of FicT toxins as T4SS substrates is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.s003" target="_blank">S3B Fig</a>. FicT of <i>M</i>. <i>extorquens</i> (UniProt identifier C7CN81 (C7CN81_METED)) is shown as a candidate T4SS substrate because it carries a BID-like sequence at its C-terminus (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.s003" target="_blank">S3C Fig</a>). (B) The genetic arrangement of <i>vbhAT</i> at the Dtr locus of pVbh of <i>B</i>. <i>schoenbuchensis</i> R1 was compared to the locus encoding the FicTA module on p1METDI of <i>M</i>. <i>extorquens</i> DM4 (genbank accession number NC_012987). (C) The illustration shows how a PezTA module on <i>Chelativorans</i> sp. BNC1 plasmid 1 (genbank accession number CP000389.1) is encoded between the conjugative VirB T4SS and its Dtr machinery in the same genetic arrangement as the VbhTA module is encoded on pVbh.</p

An integrative strategy to identify the entire protein coding potential of prokaryotic genomes by proteogenomics

Accurate annotation of all protein-coding sequences (CDSs) is an essential prerequisite to fully exploit the rapidly growing repertoire of completely sequenced prokaryotic genomes. However, large discrepancies among the number of CDSs annotated by different resources, missed functional short open reading frames (sORFs), and overprediction of spurious ORFs represent serious limitations. Our strategy toward accurate and complete genome annotation consolidates CDSs from multiple reference annotation resources, ab initio gene prediction algorithms and in silico ORFs (a modified six-frame translation considering alternative start codons) in an integrated proteogenomics database (iPtgxDB) that covers the entire protein-coding potential of a prokaryotic genome. By extending the PeptideClassifier concept of unambiguous peptides for prokaryotes, close to 95% of the identifiable peptides imply one distinct protein, largely simplifying downstream analysis. Searching a comprehensive Bartonella henselae proteomics data set against such an iPtgxDB allowed us to unambiguously identify novel ORFs uniquely predicted by each resource, including lipoproteins, differentially expressed and membrane-localized proteins, novel start sites and wrongly annotated pseudogenes. Most novelties were confirmed by targeted, parallel reaction monitoring mass spectrometry, including unique ORFs and single amino acid variations (SAAVs) identified in a re-sequenced laboratory strain that are not present in its reference genome. We demonstrate the general applicability of our strategy for genomes with varying GC content and distinct taxonomic origin. We release iPtgxDBs for B. henselae, Bradyrhizobium diazoefficiens and Escherichia coli and the software to generate both proteogenomics search databases and integrated annotation files that can be viewed in a genome browser for any prokaryote

Working model: A VbhT-like interbacterial effector as a missing link in the evolution of Beps from FicT toxins.

<p>The model illustrates the homology of genes associated with different T4SS machineries ranging from a regular conjugation system (represented by the AvhB T4SS of <i>A</i>. <i>tumefaciens</i>, bottom) to a host-targeting, effector secreting virulence factor (VirB T4SS of <i>B</i>. <i>henselae</i>, top). Core components of the T4SS machinery and the T4CP are shown in green, the Dtr machinery is shown in yellow, and TA modules or effectors are shown in red. Note that the VbhTA module of <i>B</i>. <i>schoenbuchensis</i> pVbh (second from top) displays the same domain architecture as the majority of extant Beps and likely their common ancestor. Like VbhT, the most upstream Bep (BepA) is encoded with a FicA antitoxin that is called BiaA in the context host-targeted <i>Bartonella</i> effectors [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.ref011" target="_blank">11</a>]. Based on the genomic islands with Vbh-like T4SS and FicTA modules like BtrFicTA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.g004" target="_blank">Fig 4</a>), we infer an ancient pVbh-like plasmid in which a FicTA module was encoded between the conjugative relaxase and the T4SS machinery (second from bottom). It is clearly apparent that a DNA rearrangement fusing one BID domain of the relaxase with the FicT toxin–i.e., classical terminal reassortment–would create a VbhT-like interbacterial effector as evidenced by the clear composite architecture of this protein (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.g006" target="_blank">Fig 6</a>). We therefore speculate based on these homologies that an evolutionary process from the bottom to the top of this model may approximate the evolutionary history of the host-targeting VirB T4SS.</p