Trade and socioeconomic importance of an invasive giant snail in the endemic-rich island of São Tomé, Central Africa.

Managing invasive species is crucial to mitigate their negative impacts on ecosystems, yet conflicts may arise when their social benefits are disregarded. Human pressure on the endemic-rich forests of São Tomé has been high since the island was discovered by the Portuguese in the 15th century, and numerous species have been introduced. These include the invasive West African giant land snail (Archachatina marginata), which was introduced in the mid-20th century, is now widespread on the island, and is a potential threat to native flora and fauna. We assessed the frequency of consumption of this species and its socioeconomic importance to people across the island with household questionnaires, focus group discussions, and semistructured interviews. We explored the prevalence and potential drivers of use (e.g., wealth, household composition, and diversity of occupations) and characterized the commodity chain to identify demographic groups linked to the snail trade. We interviewed 672 people (1 person per household), conducted 6 focus groups, and interviewed 80 key actors belonging to 5 subcategories. The snail was the most widely consumed bushmeat and an important source of income, particularly for women and unemployed youth. Insecure and scarce livelihood alternatives, mostly in rural areas, were reported as drivers for trade involvement. Snail harvesting was more frequent in poorer households with low occupational diversity. Selling tended to occur in households that were well-established in the community and had a higher proportion of children. Both were stimulated by the proximity of communities to the native forest. Buying snails was common in all demographic groups, but it was linked to wealth and occupational diversity. Interventions to manage the impact of this introduced species on the valuable ecosystems of the island should involve multiple sectors of society to ensure societal support. This requires robust consideration of the welfare of vulnerable demographic groups that benefit from the species.

Importancia socioeconómica y comercial de un caracol invasor en la isla rica en endemismos de Santo Tomé, África Central Resumen La gestión de las especies invasoras es crucial para mitigar sus efectos negativos en los ecosistemas, aunque pueden surgir conflictos cuando no se tienen en cuenta sus beneficios sociales. La presión humana sobre los bosques de Santo Tomé, ricos en endemismos, ha sido alta desde que la isla fue colonizada por los portugueses en el siglo XV y desde entonces se han introducido numerosas especies. Una de ellas es el caracol terrestre gigante de África Occidental (Archachatina marginata), introducido a mediados del siglo XX y que ahora tiene una distribución amplia en la isla y es una amenaza potencial para la flora y la fauna autóctonas al ser invasor. Evaluamos la frecuencia de consumo de esta especie y su importancia socioeconómica para la población de toda la isla mediante encuestas en hogares, grupos de discusión y entrevistas semiestructuradas a actores clave de cinco categorías. Exploramos la prevalencia y los posibles factores impulsores del consumo (por ejemplo, la riqueza, la composición de los hogares y la diversidad de ocupaciones) y caracterizamos la cadena comercial para identificar los grupos demográficos vinculados al comercio de caracoles. Entrevistamos a 672 personas (una persona por hogar), realizamos seis grupos de discusión y entrevistamos a 80 actores clave. El caracol fue la carne de caza más consumida y una importante fuente de ingresos, sobre todo para las mujeres y los jóvenes desempleados. La inseguridad y la escasez de medios de subsistencia, sobre todo en las zonas rurales, fueron los factores que impulsaron la participación en el mercado. La colecta de caracoles fue más frecuente en los hogares más pobres y con escasa diversidad ocupacional. La venta tendía a producirse en hogares bien establecidos en la comunidad y con una mayor proporción de niños. Ambos factores se vieron estimulados por la proximidad de las comunidades al bosque nativo. La compra de caracoles fue común en todos los grupos demográficos, pero estuvo vinculada a la riqueza y a la diversidad ocupacional. Las intervenciones para gestionar el impacto de esta especie introducida en los valiosos ecosistemas de la isla deben implicar a múltiples sectores de la sociedad para garantizar su apoyo. Para ello es necesario tener muy en cuenta el bienestar de los grupos demográficos vulnerables que se benefician de la especie.

Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis.

Peptidoglycan is the main component of the bacterial wall and protects cells from the mechanical stress that results from high intracellular turgor. Peptidoglycan biosynthesis is very similar in all bacteria; bacterial shapes are therefore mainly determined by the spatial and temporal regulation of peptidoglycan synthesis rather than by the chemical composition of peptidoglycan. The form of rod-shaped bacteria, such as Bacillus subtilis or Escherichia coli, is generated by the action of two peptidoglycan synthesis machineries that act at the septum and at the lateral wall in processes coordinated by the cytoskeletal proteins FtsZ and MreB, respectively. The tubulin homologue FtsZ is the first protein recruited to the division site, where it assembles in filaments-forming the Z ring-that undergo treadmilling and recruit later divisome proteins. The rate of treadmilling in B. subtilis controls the rates of both peptidoglycan synthesis and cell division. The actin homologue MreB forms discrete patches that move circumferentially around the cell in tracks perpendicular to the long axis of the cell, and organize the insertion of new cell wall during elongation. Cocci such as Staphylococcus aureus possess only one type of peptidoglycan synthesis machinery, which is diverted from the cell periphery to the septum in preparation for division. The molecular cue that coordinates this transition has remained elusive. Here we investigate the localization of S. aureus peptidoglycan biosynthesis proteins and show that the recruitment of the putative lipid II flippase MurJ to the septum, by the DivIB-DivIC-FtsL complex, drives peptidoglycan incorporation to the midcell. MurJ recruitment corresponds to a turning point in cytokinesis, which is slow and dependent on FtsZ treadmilling before MurJ arrival but becomes faster and independent of FtsZ treadmilling after peptidoglycan synthesis activity is directed to the septum, where it provides additional force for cell envelope constriction.

The ClpX chaperone controls autolytic splitting of Staphylococcus aureus daughter cells, but is bypassed by ß-lactam antibiotics or inhibitors of WTA biosynthesis.

ß-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of ß-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of ß-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with ß-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by ß-lactams or by inhibiting an early step in WTA biosynthesis. The finding that ß-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that ß-lactams do not kill S. aureus simply by weakening the cell wall.

The two alternative NADH:quinone oxidoreductases from Staphylococcus aureus: two players with different molecular and cellular roles.

Staphylococcus aureus is an opportunistic pathogen that has emerged as a major public health threat due to the increased incidence of its drug resistance. S. aureus presents a remarkable capacity to adapt to different niches due to the plasticity of its energy metabolism. In this work, we investigated the energy metabolism of S. aureus, focusing on the alternative NADH:quinone oxidoreductases, NDH-2s. S. aureus presents two genes encoding NDH-2s (NDH-2A and NDH-2B) and lacks genes coding for Complex I, the canonical respiratory NADH:quinone oxidoreductase. This observation makes the action of NDH-2s crucial for the regeneration of NAD+ and, consequently, for the progression of metabolism. Our study involved the comprehensive biochemical characterization of NDH-2B and the exploration of the cellular roles of NDH-2A and NDH-2B, utilizing knockout mutants (Δndh-2a and Δndh-2b). We show that NDH-2B uses NADPH instead of NADH, does not establish a charge-transfer complex in the presence of NADPH, and its reduction by this substrate is the catalytic rate-limiting step. In the case of NDH-2B, the reduction of the flavin is inherently slow, and we suggest the establishment of a charge transfer complex between NADP+ and FADH2, as previously observed for NDH-2A, to slow down quinone reduction and, consequently, prevent the overproduction of reactive oxygen species, which is potentially unnecessary. Furthermore, we observed that the lack of NDH-2A or NDH-2B impacts cell growth, volume, and division differently. The absence of these enzymes results in distinct metabolic phenotypes, emphasizing the unique cellular roles of each NDH-2 in energy metabolism.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen, posing a global challenge in clinical medicine due to the increased incidence of its drug resistance. For this reason, it is essential to explore and understand the mechanisms behind its resistance, as well as the fundamental biological features such as energy metabolism and the respective players that allow S. aureus to live and survive. Despite its prominence as a pathogen, the energy metabolism of S. aureus remains underexplored, with its respiratory enzymes often escaping thorough investigation. S. aureus bioenergetic plasticity is illustrated by its ability to use different respiratory enzymes, two of which are investigated in the present study. Understanding the metabolic adaptation strategies of S. aureus to bioenergetic challenges may pave the way for the design of therapeutic approaches that interfere with the ability of the pathogen to successfully adapt when it invades different niches within its host.

The Holliday junction resolvase RecU is required for chromosome segregation and DNA damage repair in Staphylococcus aureus.

BACKGROUND: The Staphylococcus aureus RecU protein is homologous to a Bacillus subtilis Holliday junction resolvase. Interestingly, RecU is encoded in the same operon as PBP2, a penicillin-binding protein required for cell wall synthesis and essential for the full expression of resistance in Methicillin Resistant S. aureus strains. In this work we have studied the role of RecU in the clinical pathogen S. aureus. RESULTS: Depletion of RecU in S. aureus results in the appearance of cells with compact nucleoids, septa formed over the DNA and anucleate cells. RecU-depleted cells also show increased septal recruitment of the DNA translocase SpoIIIE, presumably to resolve chromosome segregation defects. Additionally cells are more sensitive to DNA damaging agents such as mitomycin C or UV radiation. Expression of RecU from the ectopic chromosomal spa locus showed that co-expression of RecU and PBP2 was not necessary to ensure correct cell division, a process that requires tight coordination between chromosome segregation and septal cell wall synthesis. CONCLUSIONS: RecU is required for correct chromosome segregation and DNA damage repair in S. aureus. Co-expression of recU and pbp2 from the same operon is not required for normal cell division.

Detection of invasive Escherichia coli in dogs with granulomatous colitis using immunohistochemistry.

Granulomatous colitis in dogs can be associated with infection of the colonic mucosa by invasive strains of Escherichia coli. To date, fluorescence in situ hybridization (FISH) is the gold-standard method to assess intramucosal and intracellular bacterial invasion. However, FISH requires expensive fluorescence microscopy equipment and is therefore not widely available. We investigated the use of immunohistochemistry (IHC) as an alternative method to detect invasive E. coli in dogs with granulomatous colitis. Archived paraffin-embedded blocks were selected from 26 dogs with colitis, in which FISH had been performed by an outside laboratory. Using a polyclonal antibody, IHC for E. coli was performed on sections cut from the same blocks, and the presence of invasive E. coli was recorded. All 11 specimens in which FISH had detected E. coli were also positive on IHC, with strong immunolabeling in the cytoplasm of macrophages and extracellularly in the lamina propria; all 15 specimens that were negative for invasive bacteria on FISH were also negative on IHC. We found that IHC is a sensitive technique for the detection of invasive E. coli in dogs with granulomatous colitis.

Alpha-Terpineol as Antitumor Candidate in Pre-Clinical Studies.

BACKGROUND: Alpha-terpineol is monoterpene alcohol with anti-tumor activity against different tumor cell lines (lung, breast, leukemias and colorectal) through blockage of NF-kB expression, which play an important role in tumor cells growth. OBJECTIVE: Evaluate the antitumor activity of alpha-terpineol in murine Sarcoma 180 cell line. METHODS: For the tests, different cytotoxic and genotoxic assays were used, including Trypan blue, cytokinesis- blocked micronucleus assay, comet assay, agarose gel DNA fragmentation, flow cytometry and cell viability using fluorescence. Ascitic fluid cells from sarcoma 180 were obtained from Mus musculus peritoneal cavity and Alpha-terpineol was tested at 100, 250 and 500 µg/mL. Doxorubicin and Cisplatin were used as positive controls. RESULTS: Cytotoxic effects of alpha-terpineol were found in all concentrations tested, reducing cell viability in 50.9; 38.53; 30.82% at 100, 250 and 500 µg/mL, respectively. Alpha-terpineol induced genotoxic effects due to DNA fragmentation (increased frequency and index of damage), and was clastogenic by increased micronuclei formation, nucleoplasmic bridges and nuclear buds. DNA fragmentation and increased cell death indicated that alpha-terpineol can cause early, late, and necrotic apoptosis. CONCLUSION: Our data indicate that alpha-terpineol has antitumor activity revealed by cytogenetic mechanisms and / or loss of cell membrane integrity.

Reassessment of the distinctive geometry of Staphylococcus aureus cell division.

Staphylococcus aureus is generally thought to divide in three alternating orthogonal planes over three consecutive division cycles. Although this mode of division was proposed over four decades ago, the molecular mechanism that ensures this geometry of division has remained elusive. Here we show, for three different strains, that S. aureus cells do not regularly divide in three alternating perpendicular planes as previously thought. Imaging of the divisome shows that a plane of division is always perpendicular to the previous one, avoiding bisection of the nucleoid, which segregates along an axis parallel to the closing septum. However, one out of the multiple planes perpendicular to the septum which divide the cell in two identical halves can be used in daughter cells, irrespective of its orientation in relation to the penultimate division plane. Therefore, division in three orthogonal planes is not the rule in S. aureus.

SEDS-bPBP pairs direct lateral and septal peptidoglycan synthesis in Staphylococcus aureus.

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure that is essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely, through the localization of late-stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in elongation and division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 probably constitute cognate pairs of interacting proteins. A lack of RodA-PBP3 resulted in more spherical cells due to deficient sidewall PGN synthesis, whereas depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilizing the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings or arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate sidewall and septal PGN incorporation, respectively, and that their activity must be balanced to maintain coccoid morphology.

The ClpXP protease is dispensable for degradation of unfolded proteins in Staphylococcus aureus.

In living cells intracellular proteolysis is crucial for protein homeostasis, and ClpP proteases are conserved between eubacteria and the organelles of eukaryotic cells. In Staphylococcus aureus, ClpP associates to the substrate specificity factors, ClpX and ClpC forming two ClpP proteases, ClpXP and ClpCP. To address how individual ClpP proteases impact cell physiology, we constructed a S. aureus mutant expressing ClpX with an I265E substitution in the ClpP recognition tripeptide of ClpX. This mutant cannot degrade established ClpXP substrates confirming that the introduced amino acid substitution abolishes ClpXP activity. Phenotypic characterization of this mutant showed that ClpXP activity controls cell size and is required for growth at low temperature. Cells expressing the ClpXI265E variant, in contrast to cells lacking ClpP, are not sensitive to heat-stress and do not accumulate protein aggregates showing that ClpXP is dispensable for degradation of unfolded proteins in S. aureus. Consistent with this finding, transcriptomic profiling revealed strong induction of genes responding to protein folding stress in cells devoid of ClpP, but not in cells lacking only ClpXP. In the latter cells, highly upregulated loci include the urease operon, the pyrimidine biosynthesis operon, the betA-betB operon, and the pathogenicity island, SaPI5, while virulence genes were dramatically down-regulated.

FtsZ-Dependent Elongation of a Coccoid Bacterium.

UNLABELLED: A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ(G193D) allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ(G193D) filaments are more twisted and shorter than wild-type filaments. In vivo, M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE: The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

Cell shape dynamics during the staphylococcal cell cycle.

Staphylococcus aureus is an aggressive pathogen and a model organism to study cell division in sequential orthogonal planes in spherical bacteria. However, the small size of staphylococcal cells has impaired analysis of changes in morphology during the cell cycle. Here we use super-resolution microscopy and determine that S. aureus cells are not spherical throughout the cell cycle, but elongate during specific time windows, through peptidoglycan synthesis and remodelling. Both peptidoglycan hydrolysis and turgor pressure are required during division for reshaping the flat division septum into a curved surface. In this process, the septum generates less than one hemisphere of each daughter cell, a trait we show is common to other cocci. Therefore, cell surface scars of previous divisions do not divide the cells in quadrants, generating asymmetry in the daughter cells. Our results introduce a need to reassess the models for division plane selection in cocci.

Predictors of 1-year outcome after coiling for poor-grade subarachnoid aneurysmal hemorrhage.

OBJECTIVE: To describe features in patients admitted to the intensive care unit (ICU) for poor-grade aneurysmal subarachnoid hemorrhage (SAH) and to identify predictors of 12-month outcome. METHODS: We conducted a controlled observational study of 51 consecutive patients treated with endovascular coiling within 96 h of rupture for poor-grade aneurysmal SAH (20 men and 31 women, age 54 +/- 12 years). We recorded co-morbidities; initial severity; aneurysm location; occurrence of acute hydrocephalus, initial seizures, and/or neurogenic lung edema; troponin values, Fisher grade; computed tomography (CT) findings; treatment intensity; and occurrence of vasospasm. The brain injury marker S100B was assayed daily over the first 8 days. Glasgow Outcome Scores (GOS) were recorded at ICU discharge, at 6 and 12 months. The main outcome criterion was the 1-year GOS score, which we used to classify patients as having a poor outcome (GOS 1-3) or a good outcome (GOS 4-5). RESULTS: Overall, clinical status after 1 year was very good (GOS 5) in 41% of patients and good (GOS 4) in 16%. Neither baseline characteristics nor interventions differed significantly between patients with good outcome (GOS 4-5) and those with poor outcome (GOS 1-3). Persistent intracranial pressure elevation and higher mean 8-day S100B value significantly and independently predicted the 1-year GOS outcome (P = 0.008 and P = 0.001, respectively). CONCLUSIONS: Patients in poor clinical condition after SAH have more than a 50:50 chance of a favorable outcome after 1 year. High mean 8-day S100B value and persistent intracranial hypertension predict a poor outcome (GOS 1-3) after 1 year.