Sónia Castanheira

Bacterial cell division is orchestrated by proteins that assemble in dynamic complexes collectively known as the divisome. Essential monofunctional enzymes with glycosyltransferase or transpeptidase (TPase) activities, FtsW and FtsI respectively, engage in the synthesis of septal peptidoglycan (sPG). Enigmatically, Salmonella has two TPases that can promote cell division independently: FtsI (PBP3) and the pathogen‐specific paralogue PBP3_SAL. How Salmonella regulates the assembly of the sPG synthase complex with these two TPases, is unknown. Here, we characterized Salmonella division complexes in wild‐type cells and isogenic mutants lacking PBP3 or PBP3_SAL. The complexes were cross‐linked in vivo and pulled down with antibodies recognizing each enzyme. Proteomics of the immunoprecipitates showed that PBP3 and PBP3_SAL do not extensively cross‐link in wild type cells, supporting the presence of independent complexes. More than 40 proteins cross‐link in complexes in which these two TPases are present. Those identified with high scores include FtsA, FtsK, FtsQLB, FtsW, PBP1B, SPOR domain‐containing proteins (FtsN, DedD, RlpA, DamX), amidase activators (FtsX, EnvC, NlpD) and Tol‐Pal proteins. Other cross‐linked proteins are the protease Prc, the elongasome TPase PBP2 and, D,D‐endo‐ and D,D‐carboxypeptidases. PBP3 and PBP3_SAL localize at midcell and compete for occupying the division complex in response to environmental cues. Thus, a catalytic‐dead PBP3_SAL‐S300A variant impairs cell division in a high osmolarity and acidic condition in which it is produced at levels exceeding those of PBP3. Salmonella may therefore exploit an ‘adjustable’ divisome to exchange TPases for ensuring cell division in distinct environments and, in this manner, expand its colonization capacities.

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 PBP2_SAL. The PBP2-elongasome responds to neutral pH whereas that directed by PBP2_SAL assembles in acidic conditions. Moreover, the PBP2_SAL-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.

Essential peptidoglycan synthases, like penicillin binding proteins 2 and 3 (PBP2/PBP3) of Escherichia coli , define shape by orchestrating cell elongation and division, respectively. Despite being intensively studied as drug targets, the regulatory rules governing their production remain poorly understood. During infection, the closely related pathogen Salmonella enterica serovar Typhimurium downregulates PBP2/PBP3 production and replace them with alternative peptidoglycan synthases, PBP2 _SAL /PBP3 _SAL , absent in E. coli . The bases for such switch in morphogenetic proteins are unknown. Here, we show that the S. Typhimurium regulator OmpR triggers PBP2 _SAL and PBP3 _SAL expression responding solely to acid pH and define a shared motif present in upstream regions of the PBP2 _SAL- and PBP3 _SAL -coding genes sufficient for such control. The elimination of PBP2/PBP3 in infection conditions is however multifactorial, requiring acidity, high osmolarity and being favoured by OmpR and the Prc protease. Remarkably, we found that E. coli loses the essential PBP3 required for cell division when exposed to both acidity and high osmolarity, the environmental cues encountered by intracellular S. Typhimurium. Therefore, OmpR played a central role in the evolution of this pathogen when co-opting the regulation of PBP2 _SAL /PBP3 _SAL and, consequently, promoting a new morphogenetic cycle that made possible increasing progeny inside acidic eukaryotic phagosomes. Significance: Some enzymes that participate in peptidoglycan metabolism are present exclusively in bacterial pathogens and modify its structure to limit immune recognition. The intracellular pathogen Salmonella enterica serovar Typhimurium is the only example known to date in which a “substitution” of essential peptidoglycan enzymes involved in cell division and elongation takes place during infection. The data presented here support instability of PBP3 in environments with acidity and high osmolarity as a probable selective pressure that promoted the fixation of alternative morphogenetic enzymes. This was possible due to the control that OmpR exerted over these new foreign functions. The acquisition of enzymes like PBP2 _SAL and PBP3 _SAL therefore represent a “quantum leap” evolutionary event in S. Typhimurium that made possible the colonization of acidic intracellular niches.

Significance: Many phytopathogenic fungi need specialized structures, called appressoria, to penetrate the plant before infection starts. For these structures, the fungus must carefully control when and where its cells divide, and it has been reported in different fungal phytopathogens that appressorium formation is subordinated to the cell cycle control. However, the molecular details about how this control operates were unknown. We described in the corn smut fungus U. maydis that Biz1, a regulator for the formation of appressorium, is phosphorylated by the kinase responsible for cell cycle progression. Biz1 is part of a negative feedback loop responsible for the final decision to invade or not the plant tissue. Targeting this process will lead to the development of antipenetrant fungicides.

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 –

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 PBP3 _SAL —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 PBP3 _SAL or the hitherto considered essential PBP3. While PBP3 alone promotes cell division under all conditions tested, the mutant producing only PBP3 _SAL proliferates under acidic conditions (pH ≤ 5.8) but does not divide at neutral pH. PBP3 _SAL 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. PBP3 _SAL activity is also strictly dependent on acidic pH, as shown by beta-lactam antibiotic binding assays. Live-cell imaging microscopy revealed that PBP3 _SAL alone is sufficient for S . Typhimurium to divide within phagosomes of the eukaryotic cell. Additionally, we detected much larger amounts of PBP3 _SAL than those of PBP3 in vivo in bacteria colonizing mouse target organs. Therefore, PBP3 _SAL 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, PBP3 _SAL , is present only in bacterial pathogens and evolved in Salmonella to function exclusively in acidic environments. PBP3 _SAL 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.

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.