Pathogenic diversification of the gut commensal Providencia alcalifaciens via acquisition of a second type III secretion system.

Providencia alcalifaciens is a Gram-negative bacterium found in a wide variety of water and land environments and organisms. It has been isolated as part of the gut microbiome of animals and insects, as well as from stool samples of patients with diarrhea. Specific P. alcalifaciens strains encode gene homologs of virulence factors found in other pathogenic members of the same Enterobacterales order, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are also pathogenic determinants in P. alcalifaciens is not known. Here we have used P. alcalifaciens 205/92, a clinical isolate, with in vitro and in vivo infection models to investigate P. alcalifaciens -host interactions at the cellular level. Our particular focus was the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS 1b is widespread in Providencia spp. and encoded on the chromosome. T3SS 1a is encoded on a large plasmid that is present in a subset of P. alcalifaciens strains, which are primarily isolates from diarrheal patients. Using a combination of electron and fluorescence microscopy and gentamicin protection assays we show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, rapidly lyses its internalization vacuole and proliferates in the cytosol. This triggers caspase-4 dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS 1a in entry, vacuole lysis and cytosolic proliferation is host-cell type specific, playing a more prominent role in human intestinal epithelial cells as compared to macrophages. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa, inducing mild epithelial damage with negligible fluid accumulation. No overt role for T3SS 1a or T3SS 1b was seen in the calf infection model. However, T3SS 1b was required for the rapid killing of Drosophila melanogaster . We propose that the acquisition of two T3SS by horizontal gene transfer has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

MARCKS Inhibition Alters Bovine Neutrophil Responses to Salmonella Typhimurium.

Neutrophils are innate immune cells that respond quickly to sites of bacterial infection and play an essential role in host defense. Interestingly, some bacterial pathogens benefit from exuberant neutrophil inflammation. Salmonella is one such pathogen that can utilize the toxic mediators released by neutrophils to colonize the intestine and cause enterocolitis. Because neutrophils can aid gut colonization during Salmonella infection, neutrophils represent a potential host-directed therapeutic target. Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin-binding protein that plays an essential role in many neutrophil effector responses. We hypothesized that inhibition of MARCKS protein would alter bovine neutrophil responses to Salmonella Typhimurium (STm) ex vivo. We used a MARCKS inhibitor peptide to investigate the role of MARCKS in neutrophil responses to STm. This study demonstrates that MARCKS inhibition attenuated STm-induced neutrophil adhesion and chemotaxis. Interestingly, MARCKS inhibition also enhanced neutrophil phagocytosis and respiratory burst in response to STm. This is the first report describing the role of MARCKS protein in neutrophil antibacterial responses.

The Salmonella type-3 secretion system-1 and flagellar motility influence the neutrophil respiratory burst.

Neutrophils are innate immune response cells designed to kill invading microorganisms. One of the mechanisms neutrophils use to kill bacteria is generation of damaging reactive oxygen species (ROS) via the respiratory burst. However, during enteric salmonellosis, neutrophil-derived ROS actually facilitates Salmonella expansion and survival in the gut. This seeming paradox led us to hypothesize that Salmonella may possess mechanisms to influence the neutrophil respiratory burst. In this work, we used an in vitro Salmonella-neutrophil co-culture model to examine the impact of enteric infection relevant virulence factors on the respiratory burst of human neutrophils. We report that neutrophils primed with granulocyte-macrophage colony stimulating factor and suspended in serum containing complement produce a robust respiratory burst when stimulated with viable STm. The magnitude of the respiratory burst increases when STm are grown under conditions to induce the expression of the type-3 secretion system-1. STm mutants lacking the type-3 secretion system-1 induce less neutrophil ROS than the virulent WT. In addition, we demonstrate that flagellar motility is a significant agonist of the neutrophil respiratory burst. Together our data demonstrate that both the type-3 secretion system-1 and flagellar motility, which are established virulence factors in enteric salmonellosis, also appear to directly influence the magnitude of the neutrophil respiratory burst in response to STm in vitro.

Evaluation of serum amyloid A and haptoglobin concentrations as prognostic indicators for horses with colic.

OBJECTIVE: To evaluate the use of the acute-phase proteins serum amyloid A (SAA) and haptoglobin as prognostic indicators in horses with colic with regard to the need for surgical intervention, development of complications, and hospitalization cost and duration. DESIGN: Prospective observational study. ANIMALS: 20 clinically normal horses and 42 horses with colic. PROCEDURES: Total WBC and neutrophil counts and plasma fibrinogen, SAA, and haptoglobin concentrations were compared between healthy (control) horses and horses admitted to a veterinary teaching hospital for colic. Clinicopathologic values were compared between medical and surgical colic cases to test the ability of acute-phase proteins to predict indication for surgical intervention, development of complications, and duration and cost of hospitalization. RESULTS: Mean SAA concentration was significantly higher in the surgical group, compared with that for both the control and medical groups. Haptoglobin concentration did not differ significantly among groups. Horses with colic and an abnormally increased SAA concentration (> 5 µg/mL) were more likely to be managed surgically than medically (OR, 5.7; 95% confidence interval, 1.4 to 22.8). Horses with small intestinal lesions had significantly higher SAA concentrations than did control horses. Euthanasia due to a poor prognosis or the development of thrombophlebitis was more likely for horses with an SAA concentration > 5 µg/mL (OR, 7.6; 95% confidence interval, 1.1 to 52.4). A weak positive correlation (r = 0.30) was observed between cost of treatment and SAA concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Horses with colic that had an abnormally increased SAA concentration were more likely to require surgical intervention, develop thrombophlebitis, or be euthanized because of a poor prognosis despite treatment.

Evaluation of serum amyloid A and haptoglobin concentrations as prognostic indicators for horses with inflammatory disease examined at a tertiary care hospital.

OBJECTIVE: To evaluate use of serum amyloid A (SAA) and haptoglobin concentrations as prognostic indicators for horses with inflammatory disease in regard to euthanasia, complications, and hospitalization duration and cost. ANIMALS: 20 clinically normal horses and 53 horses with inflammatory disease. PROCEDURES: Total WBC count, neutrophil count, and fibrinogen, SAA, and haptoglobin concentrations were determined for clinically normal horses and horses with suspected inflammatory disease. Clinicopathologic values at admission were compared to test the use of SAA and haptoglobin concentrations in predicting euthanasia, complications, and hospitalization duration and cost. Haptoglobin and SAA concentrations of 22 horses were monitored during hospitalization to test the use of serial measurements in predicting survival and complications. RESULTS: Neutrophil count and SAA and haptoglobin concentrations were significantly different at admission for horses with inflammatory disease, compared with those for clinically normal horses. Horses with colitis and peritonitis had significantly higher SAA and haptoglobin concentrations than clinically normal horses. A moderate positive correlation (r = 0.355) between hospitalization duration and haptoglobin concentration was identified. Horses with an increase in SAA concentration between 24 and 72 hours after admission, compared with admission SAA concentration, were significantly more likely (OR, 7.0; 95% confidence interval, 1.1 to 45.9) to be euthanized or develop complications. CONCLUSIONS AND CLINICAL RELEVANCE: Concentrations of SAA and haptoglobin at admission were not significantly correlated with outcome in horses with inflammatory conditions. Acute-phase proteins likely have more utility in serial analysis rather than testing at a single time point for horses with inflammatory conditions.

Neurotropic T-cell-rich B-cell lymphoma in a 14-year-old Morgan gelding.

A 14-year-old Morgan gelding was presented for progressive weakness and muscle atrophy. The horse was initially diagnosed with equine protozoal myelitis based on history, physical examination, and laboratory diagnostics. Despite therapy, the horse declined clinically and was euthanized. Necropsy revealed a rare form of neurotropic lymphoma, described in this report.

Lymphome de cellules-B riches en cellules-T neurotropes chez un hongre Morgan âgé de 14 ans. Un hongre Morgan âgé de 14 ans a été présenté pour une faiblesse progressive et une atrophie musculaire. On a d'abord diagnostiqué la myélite protozoaire équine chez le cheval en se basant sur l'anamnèse, l'examen physique et le diagnostic en laboratoire. Malgré la thérapie, l'état clinique du cheval s'est détérioré et il a été euthanasié. La nécropsie a révélé une forme rare de lymphome neutropique, qui est décrite dans ce rapport.(Traduit par Isabelle Vallières).