Multifactor Regulation of the MdtJI Polyamine Transporter in Shigella.

The polyamine profile of Shigella, the etiological agent of bacillary dysentery in humans, differs markedly from that of E. coli, its innocuous commensal ancestor. Pathoadaptive mutations such as the loss of cadaverine and the increase of spermidine favour the full expression of the virulent phenotype of Shigella. Spermidine levels affect the expression of the MdtJI complex, a recently identified efflux pump belonging to the small multi-drug resistance family of transporters. In the present study, we have addressed the regulation of the mdtJI operon in Shigella by asking which factors influence its expression as compared to E. coli. In particular, after identifying the mdtJI promoter by primer extension analysis, in vivo transcription assays and gel-retardation experiments were carried out to get insight on the silencing of mdtJI in E. coli. The results indicate that H-NS, a major nucleoid protein, plays a key role in repressing the mdtJI operon by direct binding to the regulatory region. In the Shigella background mdtJI expression is increased by the high levels of spermidine typically found in this microorganism and by VirF, the plasmid-encoded regulator of the Shigella virulence regulatory cascade. We also show that the expression of mdtJI is stimulated by bile components. Functional analyses reveal that MdtJI is able to promote the excretion of putrescine, the spermidine precursor. This leads us to consider the MdtJI complex as a possible safety valve allowing Shigella to maintain spermidine to a level optimally suited to survival within infected macrophages and, at the same time, prevent toxicity due to spermidine over-accumulation.

Molecular and functional profiling of the polyamine content in enteroinvasive E. coli : looking into the gap between commensal E. coli and harmful Shigella.

Polyamines are small molecules associated with a wide variety of physiological functions. Bacterial pathogens have developed subtle strategies to exploit polyamines or manipulate polyamine-related processes to optimize fitness within the host. During the transition from its innocuous E. coli ancestor, Shigella, the aetiological agent of bacillary dysentery, has undergone drastic genomic rearrangements affecting the polyamine profile. A pathoadaptation process involving the speG gene and the cad operon has led to spermidine accumulation and loss of cadaverine. While a higher spermidine content promotes the survival of Shigella within infected macrophages, the lack of cadaverine boosts the pathogenic potential of the bacterium in host tissues. Enteroinvasive E. coli (EIEC) display the same pathogenicity process as Shigella, but have a higher infectious dose and a higher metabolic activity. Pathoadaption events affecting the cad locus have occurred also in EIEC, silencing cadaverine production. Since EIEC are commonly regarded as evolutionary intermediates between E. coli and Shigella, we investigated on their polyamine profile in order to better understand which changes have occurred along the path to pathogenicity. By functional and molecular analyses carried out in EIEC strains belonging to different serotypes, we show that speG has been silenced in one strain only, favouring resistance to oxidative stress conditions and survival within macrophages. At the same time, we observe that the content of spermidine and putrescine, a relevant intermediate in the synthesis of spermidine, is higher in all strains as compared to E. coli. This may represent an evolutionary response to the lack of cadaverine. Indeed, restoring cadaverine synthesis decreases the expression of the speC gene, whose product affects putrescine production. In the light of these results, we discuss the possible impact of pathoadaptation events on the evolutionary emergence of a polyamine profile favouring to the pathogenic lifestyle of Shigella and EIEC.

Polar localization of PhoN2, a periplasmic virulence-associated factor of Shigella flexneri, is required for proper IcsA exposition at the old bacterial pole.

Proper protein localization is critical for bacterial virulence. PhoN2 is a virulence-associated ATP-diphosphohydrolase (apyrase) involved in IcsA-mediated actin-based motility of S. flexneri. Herein, by analyzing a ΔphoN2 mutant of the S. flexneri strain M90T and by generating phoN2::HA fusions, we show that PhoN2, is a periplasmic protein that strictly localizes at the bacterial poles, with a strong preference for the old pole, the pole where IcsA is exposed, and that it is required for proper IcsA exposition. PhoN2-HA was found to be polarly localized both when phoN2::HA was ectopically expressed in a Escherichia coli K-12 strain and in a S. flexneri virulence plasmid-cured mutant, indicating a conserved mechanism of PhoN2 polar delivery across species and that neither IcsA nor the expression of other virulence-plasmid encoded genes are involved in this process. To assess whether PhoN2 and IcsA may interact, two-hybrid and cross-linking experiments were performed. While no evidence was found of a PhoN2-IcsA interaction, unexpectedly the outer membrane protein A (OmpA) was shown to bind PhoN2-HA through its periplasmic-exposed C-terminal domain. Therefore, to identify PhoN2 domains involved in its periplasmic polar delivery as well as in the interaction with OmpA, a deletion and a set of specific amino acid substitutions were generated. Analysis of these mutants indicated that neither the (183)PAPAP(187) motif of OmpA, nor the N-terminal polyproline (43)PPPP(46) motif and the Y155 residue of PhoN2 are involved in this interaction while P45, P46 and Y155 residues were found to be critical for the correct folding and stability of the protein. The relative rapid degradation of these amino acid-substituted recombinant proteins was found to be due to unknown S. flexneri-specific protease(s). A model depicting how the PhoN2-OmpA interaction may contribute to proper polar IcsA exposition in S. flexneri is presented.

Molecular evolution of the nicotinic acid requirement within the Shigella/EIEC pathotype.

Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor in several anabolic and catabolic reactions. NAD derives from quinolinic acid (QUIN) which in Escherichia coli is obtained through a pyridine salvage pathway or a de novo synthesis pathway. In the latter case, two enzymes, L-aspartate oxidase (NadB) and quinolinate synthase (NadA), are required for the synthesis of QUIN. In contrast to its E. coli ancestor, Shigella spp., the causative agent of bacillary dissentery, lacks the de novo pathway and strictly requires nicotinic acid for growth (Nic⁻ phenotype). This phenotype depends on the silencing of the nadB and nadA genes and its pathoadaptive nature is suggested by the observation that QUIN attenuates the Shigella invasive process. Shigella shares the pathogenicity mechanism with enteronvasive E. coli (EIEC), a group of pathogenic E. coli. On the basis of this similarity EIEC and Shigella have been grouped into a single E. coli pathotype. However EIEC strains do not constitute a homogeneous group and do not possess the complete set of characters that define Shigella strains. In this work we have analysed thirteen EIEC strains belonging to different serotypes and originating from different geographic areas. We show that, in contrast to Shigella, only some EIEC strains require nicotinic acid for growth in minimal medium. Moreover, by studying the emergence of the Nic⁻ phenotype in all serotypes of S. flexneri, as well as in S. sonnei and S. dysenteriae, we describe which molecular rearrangements occurred and which mutations are responsible for the inactivation of the nadA and nadB genes. Our data confirm that the genome of Shigella is extremely dynamic and support the hypothesis that EIEC might reflect an earlier stage of the pathoadaptation process undergone by Shigella.

Polyamines: emerging players in bacteria-host interactions.

Polyamines are small polycationic molecules found in almost all cells and associated with a wide variety of physiological processes. In recent years it has become increasingly clear that, in addition to core physiological functions, polyamines play a crucial role in bacterial pathogenesis. Considerable evidence has built up that bacteria have evolved mechanisms to turn these molecules to their own advantage and a novel standpoint to look at host-bacterium interactions emerges from the interplay among polyamines, host cells and infecting bacteria. In this review, we highlight how human bacterial pathogens have developed their own resourceful strategies to exploit polyamines or manipulate polyamine-related processes to optimize their fitness within the host. Besides contributing to a better understanding of the complex relationship between a pathogen and its host, acquisitions in this field have a significant potential towards the development of novel antibacterial therapeutic approaches.

Functional characterization of the RNA chaperone Hfq in the opportunistic human pathogen Stenotrophomonas maltophilia.

Hfq is an RNA-binding protein known to regulate a variety of cellular processes by interacting with small RNAs (sRNAs) and mRNAs in prokaryotes. Stenotrophomonas maltophilia is an important opportunistic pathogen affecting primarily hospitalized and immunocompromised hosts. We constructed an hfq deletion mutant (Δhfq) of S. maltophilia and compared the behaviors of wild-type and Δhfq S. maltophilia cells in a variety of assays. This revealed that S. maltophilia Hfq plays a role in biofilm formation and cell motility, as well as susceptibility to antimicrobial agents. Moreover, Hfq is crucial for adhesion to bronchial epithelial cells and is required for the replication of S. maltophilia in macrophages. Differential RNA sequencing analysis (dRNA-seq) of RNA isolated from S. maltophilia wild-type and Δhfq strains showed that Hfq regulates the expression of genes encoding flagellar and fimbrial components, transmembrane proteins, and enzymes involved in different metabolic pathways. Moreover, we analyzed the expression of several sRNAs identified by dRNA-seq in wild-type and Δhfq S. maltophilia cells grown in different conditions on Northern blots. The accumulation of two sRNAs was strongly reduced in the absence of Hfq. Furthermore, based on our dRNA-seq analysis we provide a genome-wide map of transcriptional start sites in S. maltophilia.

Shedding of genes that interfere with the pathogenic lifestyle: the Shigella model.

Pathoadaptive mutations are evolutionary events leading to the silencing of specific anti-virulence loci. This reshapes the core genome of a novel pathogen, adapts it to the host and boosts its harmful potential. A paradigmatic case is the emergence of Shigella, the causative agent of bacillary dysentery, from its innocuous Escherichia coli ancestor. Here we summarize current views on how pathoadaptation has allowed Shigella to progressively increase its virulence. In this context, modification of the polyamine pattern emerges as a crucial step towards full expression of the virulence program in Shigella.

A new piece of the Shigella Pathogenicity puzzle: spermidine accumulation by silencing of the speG gene [corrected].

The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation of spermidine is a key determinant in the pathogenicity strategy adopted by this microrganism.

Stenotrophomonas maltophilia strains from cystic fibrosis patients: genomic variability and molecular characterization of some virulence determinants.

The genetic relatedness of 52 Stenotrophomonas maltophilia strains, collected from various environmental and clinical sources, including cystic fibrosis (CF) patients, as well as the presence and the expression of some virulence-associated genes were studied. Pulsed-field gel electrophoresis (PFGE) analysis identified 47 profiles and three clusters of isolates with an identical PFGE pattern considered to be indistinguishable strains. Restriction fragment length polymorphism of the gyrB gene grouped the 52 strains into nine different profiles. Most CF clinical isolates (29 out of 41) showed profile 1, while the analysis of the hypervariable regions of the 16S rRNA gene revealed five distinct allelic variations, with the majority of CF isolates (23 out of 41) belonging to sequence group 1. Furthermore, the strains were characterized for motility and expression of virulence-associated genes, including genes encoding type-1 fimbriae, proteases (StmPr1 and StmPr2) and esterase. All S. maltophilia strains exhibited a very broad range of swimming and twitching motility, while none showed swarming motility. A complete smf-1 gene was PCR-amplified only from clinically derived S. maltophilia strains. Finally, the virulence of representative S. maltophilia strains impaired in the expression of proteases and esterase activities was evaluated by infecting larvae of the wax moth Galleria mellonella. The results obtained strongly indicate that the major extracellular protease StmPr1 may be a relevant virulence factor of S. maltophilia.

Interference of the CadC regulator in the arginine-dependent acid resistance system of Shigella and enteroinvasive E. coli.

A typical pathoadaptive mutation of Shigella and enteroinvasive Escherichia coli (EIEC) is the inactivation of the cad locus which comprises the genes necessary for lysine decarboxylation, an enzyme involved in pH homoeostasis. In E. coli, the cadBA operon, encoding lysine decarboxylase (CadA) and a lysine cadaverine antiporter (CadB), is submitted to the control of CadC, a positive activator whose gene maps upstream the operon, and is transcribed independently from the same strand. CadC is an integral inner membrane protein which acts both, as signal sensor and as transcriptional regulator responding to the low pH and lysine signals. Analysis of the molecular rearrangements responsible for the loss of lysine decarboxylase activity in Shigella and EIEC has revealed that the inactivation of the cadC gene is a common feature. The 3 major adaptive acid resistance (AR) systems - AR1, AR2, and AR3 - are known to be activated at low pH by Shigella and E. coli, allowing them to withstand extremely acid conditions. In this study, evaluating the survival of S. flexneri, S. sonnei, and EIEC strains complemented with a functional cadC gene and challenged at low pH, we present evidence that CadC negatively regulates the expression of the arginine-dependent adaptive acid-resistance system (AR3), encoded by the adi locus while it has no effect on the expression of AR1 and AR2 systems. Moreover, since our results indicate that in enteroinvasive strains the presence of CadC reduces the expression of the arginine decarboxylase encoding gene adiA, it is possible to hypothesize that the loss of functionality of lysine decarboxylase is counterbalanced by a higher expression of the adi system, and that CadC, besides specifically affecting the regulation of the cadBA operon, is also relevant to other systems responding to low pH.

PCR-based rapid genotyping of Stenotrophomonas maltophilia isolates.

BACKGROUND: All bacterial genomes contain repetitive sequences which are members of specific DNA families. Such repeats may occur as single units, or found clustered in multiple copies in a head-to-tail configuration at specific loci. The number of clustered units per locus is a strain-defining parameter. Assessing the length variability of clusters of repeats is a versatile typing methodology known as multilocus variable number of tandem repeat analysis (MLVA). RESULTS: Stenotrophomonas maltophilia is an environmental bacterium increasingly involved in nosocomial infections and resistant to most antibiotics. The availability of the whole DNA sequence of the S. maltophilia strain K279a allowed us to set up fast and accurate PCR-based diagnostic protocols based on the measurement of length variations of loci carrying a variable number of short palindromic repeats marking the S. maltophilia genome. On the basis of the amplimers size, it was possible to deduce the number of repeats present at 12 different loci in a collection of S. maltophilia isolates, and therefore label each of them with a digit. PCR-negative regions were labelled 0. Co-amplification of two pairs of loci provided a 4-digit code sufficient for immediate subtyping. By increasing the number of loci analyzed, it should be possible to assign a more specific digit profile to isolates. In general, MLVA data match genotyping data obtained by PFGE (pulsed-field gel electrophoresis). However, some isolates exhibiting the same PCR profiles at all loci display distinct PFGE patterns. CONCLUSION: The utilization of the present protocol allows to type several S. maltophilia isolates in hours. The results are immediately interpretable without the need for sophisticated softwares. The data can be easily reproducible, and compared among different laboratories.

The two-faced role of cad genes in the virulence of pathogenic Escherichia coli.

In enterobacteria, acid stress induces expression of the cad system which is involved in maintaining intracellular pH at levels compatible with cell survival. Despite its crucial role, the cad operon is silenced in Shigella and in other pathogenic Escherichia coli. In the present review, we will address the question of why and how the cad locus has been sacrificed for the sake of optimal expression of virulence traits.

Plasticity of the P junc promoter of ISEc11, a new insertion sequence of the IS1111 family.

We describe identification and functional characterization of ISEc11, a new insertion sequence that is widespread in enteroinvasive E. coli (EIEC), in which it is always present on the virulence plasmid (pINV) and very frequently also present on the chromosome. ISEc11 is flanked by subterminal 13-bp inverted repeats (IRs) and is bounded by 3-bp terminal sequences, and it transposes with target specificity without generating duplication of the target site. ISEc11 is characterized by an atypical transposase containing the DEDD motif of the Piv/MooV family of DNA recombinases, and it is closely related to the IS1111 family. Transposition occurs by formation of minicircles through joining of the abutted ends and results in assembly of a junction promoter (P juncC) containing a -10 box in the interstitial sequence and a -35 box upstream of the right IR. A natural variant of ISEc11 (ISEc11p), found on EIEC pINV plasmids, contains a perfect duplication of the outermost 39 bp of the right end. Upon circularization, ISEc11p forms a junction promoter (P juncP) which, despite carrying -10 and -35 boxes identical to those of P juncC, exhibits 30-fold-greater strength in vivo. The discovery of only one starting point in primer extension experiments rules out the possibility that there are alternative promoter sites within the 39-bp duplication. Analysis of in vitro-generated transcripts confirmed that at limiting RNA polymerase concentrations, the activity of P juncP is 20-fold higher than the activity of P juncC. These observations suggest that the 39-bp duplication might host cis-acting elements that facilitate the binding of RNA polymerase to the promoter.

Molecular evolution of the lysine decarboxylase-defective phenotype in Shigella sonnei.

Shigella, the etiological agent of the bacillary dysentery, belongs to the extremely diverse species of Escherichia coli. In the evolutionary route of Shigella from commensal E. coli ancestors towards a pathogenic lifestyle, the critical events have been the acquisition of the pINV plasmid, through horizontal transfer and the inactivation of pre-existing genes. These so-called pathoadaptive mutations affect the expression of genes negatively interfering with the newly acquired functions necessary for the colonization of the host niche. Cadaverine, a small polyamine resulting from decarboxylation of lysine, has been shown to hamper the full expression of Shigella invasiveness mainly by altering the inflammatory response. Recent analysis of the evolution of the Shigella and enteroinvasive E. coli (EIEC) cad region indicates that silencing of the cad locus has been attained with several strategies. The increasing relevance of S. sonnei in both, developing and industrial countries, prompted us to analyze the molecular origin of the LDC- phenotype in these strains. The results obtained on several S. sonnei strains reveal that despite the difference in geographic origin and antibiotic resistance patterns, all the strains have undergone the same modifications. Multiple IS insertions into the cadBA operon have interrupted gene continuity without inducing deletions or inversions of the cadA and cadB genes which are remained entirely conserved. Moreover, by functional analysis we show that all the strains carry a defective cadC gene, thus strengthening the hypothesis that inactivation of the regulatory cadC gene might have been the first step towards a complete lack of the cad locus.

CadC is the preferential target of a convergent evolution driving enteroinvasive Escherichia coli toward a lysine decarboxylase-defective phenotype.

Enteroinvasive E. coli (EIEC), like Shigella, is the etiological agent of bacillary dysentery, a particularly severe syndrome in children in developing countries. All EIEC strains share with Shigella the inability to synthesize lysine decarboxylase (the LDC phenotype). The lack of this function is considered a pathoadaptive mutation whose emergence was necessary to obtain the full expression of invasiveness. Cadaverine, the product of lysine decarboxylation, is a small polyamine which interferes mainly with the inflammatory process induced by dysenteric bacteria. Genes coding for lysine decarboxylase and its transporter constitute a single operon (cadBA) and are expressed at low pH under the positive control of CadC. This regulator is an inner membrane protein that is able to sense pH variation and to respond by transcriptionally activating the cadBA genes. In this study we show that, unlike in Shigella, mutations affecting the cad locus in the EIEC strains we have analyzed are not followed by a novel gene arrangement and that the LCD(-) phenotype is dependent mainly on inactivation of the cadC gene. Introduction of a functional CadC restores cadaverine expression in all EIEC strains harboring either an IS2 element or a defective cadC promoter. Comparative analysis between the cad regions of S. flexneri and EIEC suggests that the LDC(-) phenotype has been attained by different strategies within the E. coli species.

Histone-like proteins and the Shigella invasivity regulon.

The contribution of histone-like proteins to the transcriptional regulation of virulence gene networks is a common feature among pathogenic bacteria. In this article we review current knowledge about the regulative role of major histone-like proteins in the silencing/activation of the invasivity phenotype of Shigella, the etiological agent of bacillary dissentery.

Enteroinvasive Escherichia coli virulence-plasmid-carried apyrase (apy) and ospB genes are organized as a bicistronic operon and are subject to differential expression.

In Shigella flexneri and enteroinvasive Escherichia coli (EIEC) the expression of the virulence-plasmid(pINV)-carried potential pathogenesis-associated apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated by the same regulators that govern the expression of virulence genes. To understand the transcriptional organization of the apy gene, the authors sequenced an 8023 bp PstI fragment of the pINV of EIEC strain HN280, which encompasses apy as well as its adjacent genes. The PstI fragment displays 99% identity with the corresponding fragment of pWR100, the pINV of S. flexneri strain M90T, and contains four genes. One of these genes, ospB, encodes a secreted protein of unknown activity and is located immediately upstream of apy. Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that ospB and apy are co-transcribed as a 2 kb bicistronic, temperature-regulated mRNA from an upstream promoter that precedes ospB. The 2 kb mRNA is post-transcriptionally processed in the intercistronic ospB-apy region, leading to the considerable accumulation of a more stable 1 kb apy-specific mRNA (half-life of 2.2+/-0.3 min, versus 27+/-4 s for the 2 kb transcript). Upon temperature induction, peak expression of the ospB-apy operon occurs when bacteria enter into the late phases of bacterial growth, where the apy-specific transcript was found to be much more prevalent if compared to the ospB-apy transcript.