Evidence of two differentially regulated elongasomes in Salmonella.

Cell shape is genetically inherited by all forms of life. Some unicellular microbes increase niche adaptation altering shape whereas most show invariant morphology. A universal system of peptidoglycan synthases guided by cytoskeletal scaffolds defines bacterial shape. In rod-shaped bacteria, this system consists of two supramolecular complexes, the elongasome and divisome, which insert cell wall material along major and minor axes. Microbes with invariant shape are thought to use a single morphogenetic system irrespective of the occupied niche. Here, we provide evidence for two elongasomes that generate (rod) shape in the same bacterium. This phenomenon was unveiled in Salmonella, a pathogen that switches between extra- and intracellular lifestyles. The two elongasomes can be purified independently, respond to different environmental cues, and are directed by distinct peptidoglycan synthases: the canonical PBP2 and the pathogen-specific homologue PBP2SAL. The PBP2-elongasome responds to neutral pH whereas that directed by PBP2SAL assembles in acidic conditions. Moreover, the PBP2SAL-elongasome moves at a lower speed. Besides Salmonella, other human, animal, and plant pathogens encode alternative PBPs with predicted morphogenetic functions. Therefore, contrasting the view of morphological plasticity facilitating niche adaptation, some pathogens may have acquired alternative systems to preserve their shape in the host.

Affinity of cefotiam for the alternative penicillin binding protein PBP3SAL used by Salmonella inside host eukaryotic cells.

BACKGROUND: Following the invasion of eukaryotic cells, Salmonella enterica serovar Typhimurium replaces PBP2/PBP3, main targets of ß-lactam antibiotics, with PBP2SAL/PBP3SAL, two homologue peptidoglycan synthases absent in Escherichia coli. PBP3SAL promotes pathogen cell division in acidic environments independently of PBP3 and shows low affinity for ß-lactams that bind to PBP3 such as aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, cefuroxime and cefalotin. OBJECTIVES: To find compounds with high affinity for PBP3SAL to control Salmonella intracellular infections. METHODS: An S. Typhimurium ΔPBP3 mutant that divides using PBP3SAL and its parental wild-type strain, were exposed to a library of 1520 approved drugs in acidified (pH 4.6) nutrient-rich LB medium. Changes in optical density associated with cell filamentation, a read-out of blockage in cell division, were monitored. Compounds causing filamentation in the ΔPBP3 mutant but not in wild-type strain-the latter strain expressing both PBP3 and PBP3SAL in LB pH 4.6-were selected for further study. The bactericidal effect due to PBP3SAL inhibition was evaluated in vitro using a bacterial infection model of cultured fibroblasts. RESULTS: The cephalosporin cefotiam exhibited higher affinity for PBP3SAL than for PBP3 in bacteria growing in acidified LB pH 4.6 medium. Cefotiam also proved to be effective against intracellular Salmonella in a PBP3SAL-dependent manner. Conversely, cefuroxime, which has higher affinity for PBP3, showed decreased effectiveness in killing intracellular Salmonella. CONCLUSIONS: Antibiotics with affinity for PBP3SAL, like the cephalosporin cefotiam, have therapeutic value for treating Salmonella intracellular infections.

OmpR and Prc contribute to switch the Salmonella morphogenetic program in response to phagosome cues.

Salmonella enterica serovar Typhimurium infects eukaryotic cells residing within membrane-bound phagosomes. In this compartment, the pathogen replaces the morphogenetic penicillin-binding proteins 2 and 3 (PBP2/PBP3) with PBP2SAL /PBP3SAL , two proteins absent in Escherichia coli. The basis for this switch is unknown. Here, we show that PBP3 protein levels drop drastically when S. Typhimurium senses acidity, high osmolarity and nutrient scarcity, cues that activate virulence functions required for intra-phagosomal survival and proliferation. The protease Prc and the transcriptional regulator OmpR contribute to lower PBP3 levels whereas OmpR stimulates PBP2SAL /PBP3SAL production. Surprisingly, despite being essential for division in E. coli, PBP3 levels also drop in non-pathogenic and pathogenic E. coli exposed to phagosome cues. Such exposure alters E. coli morphology resulting in very long bent and twisted filaments indicative of failure in the cell division and elongation machineries. None of these aberrant shapes are detected in S. Typhimurium. Expression of PBP3SAL restores cell division in E. coli exposed to phagosome cues although the cells retain elongation defects in the longitudinal axis. By switching the morphogenetic program, OmpR and Prc allow S. Typhimurium to properly divide and elongate inside acidic phagosomes maintaining its cellular dimensions and the rod shape.

Peptidoglycan editing in non-proliferating intracellular Salmonella as source of interference with immune signaling.

Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan purified from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropeptides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)-meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This peptidoglycan has a reduced glycan chain average length and ~30% increase in the L,D-crosslink, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D-transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L,D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhimurium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remarkably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-containing muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D-alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells.

A Novel Salmonella Periplasmic Protein Controlling Cell Wall Homeostasis and Virulence.

Horizontal gene transfer has shaped the evolution of Salmonella enterica as pathogen. Some functions acquired by this mechanism include enzymes involved in peptidoglycan (PG) synthesis and remodeling. Here, we report a novel serovar Typhimurium protein that is absent in non-pathogenic bacteria and bears a LprI functional domain, first reported in a Mycobacterium tuberculosis lipoprotein conferring lysozyme resistance. Based on the presence of such domain, we hypothesized a role of this S. Typhimurium protein in PG metabolism. This protein, which we named ScwA for Salmonella cell wall-related regulator-A, controls positively the levels of the murein lytic transglycosylase MltD. In addition, the levels of other enzymes that cleave bonds in the PG lattice were affected in a mutant lacking ScwA, including a soluble lytic tranglycosylase (Slt), the amidase AmiC, and a few endo- and carboxypeptidases (NlpC, PBP4, and AmpH). The scwA gene has lower G+C content than the genomic average (43.1 vs. 52.2%), supporting acquisition by horizontal transfer. ScwA is located in the periplasm, stabilized by two disulfide bridges, produced preferentially in stationary phase and down-regulated following entry of the pathogen into eukaryotic cells. ScwA deficiency, however, results in a hypervirulent phenotype in the murine typhoid model. Based on these findings, we conclude that ScwA may be exploited by S. Typhimurium to ensure cell envelope homeostasis along the infection and to prevent host overt damage. This role could be accomplished by controlling the production or stability of a reduced number of peptidoglycan hydrolases whose activities result in the release of PG fragments.

Incompatibility between proliferation and plant invasion is mediated by a regulator of appressorium formation in the corn smut fungus Ustilago maydis.

Plant pathogenic fungi often developed specialized infection structures to breach the outer surface of a host plant. These structures, called appressoria, lead the invasion of the plant by the fungal hyphae. Studies in different phytopathogenic fungi showed that appressorium formation seems to be subordinated to the cell cycle. This subordination ensures the loading in the invading hypha of the correct genetic information to proceed with plant infection. However, how the cell cycle transmits its condition to the genetic program controlling appressorium formation and promoting the plant's invasion is unknown. Our results have uncovered how this process occurs for the appressorium of Ustilago maydis, the agent responsible for corn smut disease. Here, we described that the complex Clb2-cyclin-dependent kinase (Cdk)1, one of the master regulators of G2/M cell cycle progression in U. maydis, interacts and controls the subcellular localization of Biz1, a transcriptional factor required for the activation of the appressorium formation. Besides, Biz1 can arrest the cell cycle by down-regulation of the gene encoding a second b-cyclin Clb1 also required for the G2/M transition. These results revealed a negative feedback loop between appressorium formation and cell cycle progression in U. maydis, which serves as a "toggle switch" to control the fungal decision between infecting the plant or proliferating out of the plant.

An alternative penicillin-binding protein involved in Salmonella relapses following ceftriaxone therapy.

BACKGROUND: Salmonella causes intracellular infections in humans. Besides quinolones, third generation cephalosporins are first line drugs used for salmonellosis therapy. An unresolved anomaly of this practice involves high relapse rates associated to quinolone- or cephalosporin-susceptible Salmonella isolates in patients that are discharged clinically following initial recovery. Reduced drug accessibility to intracellular locations has been hypothesized to impair pathogen eradication although supporting evidence is lacking in vivo. Here, we uncover a novel penicillin-binding protein as the first Salmonella factor likely contributing to relapse following beta-lactam, mainly ceftriaxone, therapy. METHODS: We used Salmonella enterica serovar Typhimurium mutants lacking the alternative penicillin-binding proteins PBP2SAL or PBP3SAL. Affinity of PBP2SAL and PBP3SAL for beta-lactam antibiotics was tested. Relapse after ceftriaxone therapy was analysed in the murine typhoid model. FINDINGS: S. Typhimurium does not express PBP2SAL or PBP3SAL in the Mueller-Hinton medium used for susceptibility testing. The pathogen produces these PBPs in response to acidic pH and nutrient limitation, conditions found in phagosomes of mammalian cells. PBP3SAL has low affinity for beta-lactams, even at acidic pH. In vitro susceptibility to ceftriaxone at low pH is strongly reduced. S. Typhimurium lacking PBP3SAL was unable to cause relapse in mice following ceftriaxone therapy. INTERPRETATION: The reduced capacity of ceftriaxone to clear S. Typhimurium in vivo is favoured by a switch in beta-lactam targets. This switch, involving production of the less-susceptible PBP3SAL, remains invisible for standard procedures used in clinical therapy. We conclude that eradication of salmonellosis will be possible only upon targeting of PBP3SAL with novel drugs.

Cytoplasmic retention and degradation of a mitotic inducer enable plant infection by a pathogenic fungus.

In the fungus Ustilago maydis, sexual pheromones elicit mating resulting in an infective filament able to infect corn plants. Along this process a G2 cell cycle arrest is mandatory. Such as cell cycle arrest is initiated upon the pheromone recognition in each mating partner, and sustained once cell fusion occurred until the fungus enter the plant tissue. We describe that the initial cell cycle arrest resulted from inhibition of the nuclear transport of the mitotic inducer Cdc25 by targeting its importin, Kap123. Near cell fusion to take place, the increase on pheromone signaling promotes Cdc25 degradation, which seems to be important to ensure the maintenance of the G2 cell cycle arrest to lead the formation of the infective filament. This way, premating cell cycle arrest is linked to the subsequent steps required for establishment of the infection. Disabling this connection resulted in the inability of fungal cells to infect plants.

Two distinct penicillin binding proteins promote cell division in different Salmonella lifestyles.

The bacterial cell wall preserves cell integrity in response to external insults and the internal turgor pressure. The major component of the cell wall is the peptidoglycan (PG); a giant macromolecule formed by glycan chains cross-linked by short peptides. The PG is synthesized by a stepwise process that includes cytosolic and periplasmic reactions. The building subunits -muropeptides- are incorporated into the growing macromolecule by transglycolyslation (TG) and transpeptidation (TP) reactions, which constitute the last biosynthetic steps. TP reactions, involving cleavage of the terminal D Ala-D-Ala bond in the stem peptide, are carried out by enzymes known generically as penicillin-binding proteins (PBPs) due to their capacity to bind ß lactam antibiotics, which are D Ala-D-Ala structural analogues. On an average, bacterial genomes harbour a minimum of 10 PBP-encoding genes, most of them non-essential. This dispensability has led to the widely accepted concept of functional redundancy for many PBPs. An exemption is the PBP dedicated to build the septal PG required to separate daughter cells during cell division. To date, this division specific PBP was reported as unique in all known bacteria and, as a consequence, "essential". Our recent results obtained in the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium challenges this view since this bacterium has two PBPs that can independently build the division septum. One of these two division PG enzymes is orthologue of the division-specific PBP3 of Escherichia coli. The second enzyme, named PBP3SAL, is absent in non-pathogenic bacteria and, at least in S. Typhimurium, displays PG biosynthetic activity restricted to acidic conditions. Our work also revealed that it is possible to generate a S. Typhimurium mutant defective in PBP3, which cannot divide at neutral pH.

A Specialized Peptidoglycan Synthase Promotes Salmonella Cell Division inside Host Cells.

Bacterial cell division has been studied extensively under laboratory conditions. Despite being a key event in the bacterial cell cycle, cell division has not been explored in vivo in bacterial pathogens interacting with their hosts. We discovered in Salmonella enterica serovar Typhimurium a gene absent in nonpathogenic bacteria and encoding a peptidoglycan synthase with 63% identity to penicillin-binding protein 3 (PBP3). PBP3 is an essential cell division-specific peptidoglycan synthase that builds the septum required to separate daughter cells. Since S Typhimurium carries genes that encode a PBP3 paralog-which we named PBP3SAL-and PBP3, we hypothesized that there are different cell division events in host and nonhost environments. To test this, we generated S Typhimurium isogenic mutants lacking PBP3SAL or the hitherto considered essential PBP3. While PBP3 alone promotes cell division under all conditions tested, the mutant producing only PBP3SAL proliferates under acidic conditions (pH ≤ 5.8) but does not divide at neutral pH. PBP3SAL production is tightly regulated with increased levels as bacteria grow in media acidified up to pH 4.0 and in intracellular bacteria infecting eukaryotic cells. PBP3SAL activity is also strictly dependent on acidic pH, as shown by beta-lactam antibiotic binding assays. Live-cell imaging microscopy revealed that PBP3SAL alone is sufficient for S Typhimurium to divide within phagosomes of the eukaryotic cell. Additionally, we detected much larger amounts of PBP3SAL than those of PBP3 in vivo in bacteria colonizing mouse target organs. Therefore, PBP3SAL evolved in S Typhimurium as a specialized peptidoglycan synthase promoting cell division in the acidic intraphagosomal environment.IMPORTANCE During bacterial cell division, daughter cells separate by a transversal structure known as the division septum. The septum is a continuum of the cell wall and therefore is composed of membrane(s) and a peptidoglycan layer. To date, actively growing bacteria were reported to have only a "cell division-specific" peptidoglycan synthase required for the last steps of septum formation and consequently, essential for bacterial life. Here, we discovered that Salmonella enterica has two peptidoglycan synthases capable of synthesizing the division septum. One of these enzymes, PBP3SAL, is present only in bacterial pathogens and evolved in Salmonella to function exclusively in acidic environments. PBP3SAL is used preferentially by Salmonella to promote cell division in vivo in mouse target organs and inside acidified phagosomes. Our data challenge the concept of only one essential cell division-specific peptidoglycan synthase and demonstrate that pathogens can divide in defined host locations using alternative mechanisms.

Salmonella Populations inside Host Cells.

Bacteria of the Salmonella genus cause diseases ranging from gastroenteritis to life-threatening typhoid fever and are among the most successful intracellular pathogens known. After the invasion of the eukaryotic cell, Salmonella exhibits contrasting lifestyles with different replication rates and subcellular locations. Although Salmonella hyper-replicates in the cytosol of certain host cell types, most invading bacteria remain within vacuoles in which the pathogen proliferates at moderate rates or persists in a dormant-like state. Remarkably, these cytosolic and intra-vacuolar intracellular lifestyles are not mutually exclusive and can co-exist in the same infected host cell. The mechanisms that direct the invading bacterium to follow the cytosolic or intra-vacuolar "pathway" remain poorly understood. In vitro studies show predominance of either the cytosolic or the intra-vacuolar population depending on the host cell type invaded by the pathogen. The host and pathogen factors controlling phagosomal membrane integrity and, as consequence, the egress into the cytosol, are intensively investigated. Other aspects of major interest are the host defenses that may affect differentially the cytosolic and intra-vacuolar populations and the strategies used by the pathogen to circumvent these attacks. Here, we summarize current knowledge about these Salmonella intracellular subpopulations and discuss how they emerge during the interaction of this pathogen with the eukaryotic cell.

Virulence-specific cell cycle and morphogenesis connections in pathogenic fungi.

To initiate pathogenic development, pathogenic fungi respond to a set of inductive cues. Some of them are of an extracellular nature (environmental signals), while others are intracellular (developmental signals). These signals must be integrated into a single response whose major outcome is changes in the morphogenesis of the fungus. The regulation of the cell cycle is pivotal during these cellular differentiation steps; therefore, cell cycle regulation would likely provide control points for infectious development by fungal pathogens. Here, we provide clues to understanding how the control of the cell cycle is integrated with the morphogenesis program in pathogenic fungi, and we review current examples that support these connections.

Appressorium formation in the corn smut fungus Ustilago maydis requires a G2 cell cycle arrest.

Many of the most important plant diseases are caused by fungal pathogens that form specialized cell structures to breach the leaf surface as well as to proliferate inside the plant. To initiate pathogenic development, the fungus responds to a set of inductive cues. Some of them are of extracellular nature (environmental signals) while others respond to intracellular conditions (developmental signals). These signals have to be integrated into a single response that has as a major outcome changes in the morphogenesis of the fungus. The cell cycle regulation is pivotal during these cellular differentiations, and we hypothesized that cell cycle regulation would be likely to provide control points for infection development by fungal pathogens. Although efforts have been done in various fungal systems, there is still limited information available regarding the relationship of these processes with the induction of the virulence programs. Hence, the role of fungal cell cycle regulators -which are wide conserved elements- as true virulence factors, has yet to be defined. Here we discuss the recent finding that the formation of the appressorium, a structure required for plant penetration, in the corn smut fungus Ustilago maydis seems to be incompatible with an active cell cycle and, therefore genetic circuits evolved in this fungus to arrest the cell cycle during the growth of this fungus on plant surface, before the appressorium-mediated penetration into the plant tissue.

Programmed cell cycle arrest is required for infection of corn plants by the fungus Ustilago maydis.

Ustilago maydis is a plant pathogen that requires a specific structure called infective filament to penetrate the plant tissue. Although able to grow, this filament is cell cycle arrested on the plant surface. This cell cycle arrest is released once the filament penetrates the plant tissue. The reasons and mechanisms for this cell cycle arrest are unknown. Here, we have tried to address these questions. We reached three conclusions from our studies. First, the observed cell cycle arrest is the result of the cooperation of at least two distinct mechanisms: one involving the activation of the DNA damage response (DDR) cascade; and the other relying on the transcriptional downregulation of Hsl1, a kinase that modulates the G2/M transition. Second, a sustained cell cycle arrest during the infective filament step is necessary for the virulence in U. maydis, as a strain unable to arrest the cell cycle was severely impaired in its ability to infect corn plants. Third, production of the appressorium, a structure required for plant penetration, is incompatible with an active cell cycle. The inability to infect plants by strains defective in cell cycle arrest seems to be caused by their failure to induce the appressorium formation process. In summary, our findings uncover genetic circuits to arrest the cell cycle during the growth of this fungus on the plant surface, thus allowing the penetration into plant tissue.

Fungal model systems and the elucidation of pathogenicity determinants.

Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity.

O criterio metodologico para avaliar pesquisas na area de autocontrole

Este artigo revisa a literatura sobre autocontrole indexada no Pyschological Abstracts, no periodo de 1987 a 1991, com base em duas das sete dimensoes de Baer, Wolf & Risley (1968, 1987), criadas para carcterizar e diferenciar um estudo de analise do comportamento aplicada de um estudo esperimental de laboratorio, e em duas das subcategorias de Hayes, Rincovr E Solnick (1980), contruidas para identificar as tendencias dos trabalhos nessa area de conhecimento, na decada de 70, e avaliar as direcoes assumidas no campo de aplicacao. Considerando-se a relevancia da analise metodologica e sua relacao com a producao de conhecimento, este trabalho prioriza o criterio metodologico, discutindo seu significado e suas consequencias para o direcionamento da pesquisa na area do autocontrole, especialmente quanto a escolha do problema a ser investigado e as condicoes de aplicacao.

O criterio metodologico para avaliar pesquisas na area de autocontrole

Este artigo revisa a literatura sobre autocontrole indexada no Psychological Abstracts, no periodo de 1987 a 1991, com base em duas das sete dimensoes de BAER, WOLF e RISLEY (1968, 1987), criadas para caracterizar e diferenciar um estudo de analise do comportamento aplicada de um estudo experimental de laboratorio, e em duas das subcategorias de HAYES, RINCOVER e SOLNICK (1980), construidas para identificar as tendencias dos trabalhos nessa area de conhecimento, na decada de 70, e avaliar as direcoes assumidas no campo de aplicacao. Considerando-se a relevancia da analise metodologica e sua relacao com a producao de conhecimento, este trabalho prioriza o criterio metodologico, discutindo seu significado e suas consequencias para o direcionamento da pesquisa na area do autocontrole, especialmente quanto a escolha do problema a ser investigado e as condicoes de aplicacao.