Salmonella Typhimurium employs spermidine to exert protection against ROS-mediated cytotoxicity and rewires host polyamine metabolism to ameliorate its survival in macrophages.

Salmonella infection entails a cascade of attacks and defence measures. After breaching the intestinal epithelial barrier, Salmonella is phagocytosed by macrophages, where the bacteria encounter multiple stresses, to which it employs relevant countermeasures. Our study shows that, in Salmonella, the polyamine spermidine activates a stress response mechanism by regulating critical antioxidant genes. Salmonella Typhimurium mutants for spermidine transport and synthesis cannot mount an antioxidative response, resulting in high intracellular ROS levels. These mutants are also compromised in their ability to be phagocytosed by macrophages. Furthermore, it regulates a novel enzyme in Salmonella, Glutathionyl-spermidine synthetase (GspSA), which prevents the oxidation of proteins in E. coli. Moreover, the spermidine mutants and the GspSA mutant show significantly reduced survival in the presence of hydrogen peroxide in vitro and reduced organ burden in the mouse model of Salmonella infection. Conversely, in macrophages isolated from gp91phox-/- mice, we observed a rescue in the attenuated fold proliferation previously observed upon infection. We found that Salmonella upregulates polyamine biosynthesis in the host through its effectors from SPI-1 and SPI-2, which addresses the attenuated proliferation observed in spermidine transport mutants. Thus, inhibition of this pathway in the host abrogates the proliferation of Salmonella Typhimurium in macrophages. From a therapeutic perspective, inhibiting host polyamine biosynthesis using an FDA-approved chemopreventive drug, D, L-α-difluoromethylornithine (DFMO), reduces Salmonella colonisation and tissue damage in the mouse model of infection while enhancing the survival of infected mice. Therefore, our work provides a mechanistic insight into the critical role of spermidine in stress resistance of Salmonella. It also reveals a bacterial strategy in modulating host metabolism to promote their intracellular survival and shows the potential of DFMO to curb Salmonella infection.

Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Clinical and microbiological characterization of Salmonella spp. isolates from patients treated in a university hospital in South America between 2012-2021: a cohort study.

BACKGROUND: Salmonellosis is a major cause of morbidity and mortality and one of the most frequent etiologies of diarrhea in the world. Mortality due to Salmonellosis in Latin America still poorly understood, and there is a lack of studies that evaluate resistance and clinical manifestations. The aims of this study were to characterize patients infected with Salmonella spp. seen in a university hospital in Colombia between 2012 and 2021, to evaluate trends in antibiotic resistance and to determine the proportion of overall mortality and related factors. METHODS: Retrospective observational study. All patients with microbiological diagnosis of Salmonella spp. were included. The sociodemographic, clinical and microbiological characteristics were described, and the proportion of antibiotic resistant isolates per year was estimated. The prevalence of mortality according to age groups was calculated. Log binomial regression models were used to establish factors associated with mortality. RESULTS: Five hundred twenty-two patients were analyzed. Salmonellosis accounted for 0.01% of all medical consultations. The median age was 16 years old. The most common clinical presentation was gastroenteric syndrome (77.1%) and symptoms included diarrhea (79.1%), fever (66.7%), abdominal pain (39.6%) and vomiting (35.2%). Of the Salmonella spp. isolates, 78.2% were not classified, 19.1% corresponded to non-typhoidal Salmonella and 2.7% to Salmonella typhi. Mortality occurs in 4.02% of the patients and was higher in patients with hematologic malignancy (11.6%). When analyzing by age group, the proportion of deaths was 2.8% in patients aged 15 years or younger, while in those older than 15 years it was 5.4%. Factors associated to mortality where bacteremia (aPR = 3.41 CI95%: 1.08-10.76) and to require treatment in the ICU (aPR = 8.13 CI95%: 1.82-37.76). In the last 10 years there has been a steady increase in resistance rates to ciprofloxacin, ampicillin, ampicillin/sulbactam and ceftriaxone, reaching rates above 60% in recent years. CONCLUSIONS: Despite improved availability of antibiotics for the treatment of salmonellosis in the past decades, mortality due to salmonellosis continues occurring in children and adults, mainly in patients with hematological malignancies and bacteremia. Antibiotic resistance rates have increased significantly over the last 10 years. Public health strategies for the control of this disease should be strengthened, especially in vulnerable populations.

[Clinical features and antimicrobial resistance of invasive non-typhoid Salmonella infection in children at Xiamen].

Objective: To investigate the clinical characteristics, serogroups and antimicrobial resistance of invasive non-typhoid Salmonella infection in children at Xiamen. Methods: Retrospective cohort study. The clinical manifestations, treatment, prognosis, serogroups and antimicrobial resistance of 29 hospitalized children with invasive non-typhoid Salmonella infection confirmed by blood, cerebrospinal fluid, bone marrow and other sterile body fluids or deep pus culture at the Department of Infectious Diseases, the Department of Orthopedics and the Department of General Surgery in Xiamen Children's Hospital from January 2016 to December 2021 were analyzed. According to the clinical diagnosis criteria, the patients were divided into sepsis group and non-sepsis group (bacteremia and local suppurative infection). The inflammatory markers, serogroups distribution and drug resistance were compared between the two groups. Comparison between groups using Mann-Whitney U test and χ2 test. Results: Among the 29 cases, there were 17 males and 12 females, with an onset age of 14 (9, 25) months, and 10 cases (34%) of patients were younger than 1 year old, 15 cases (52%) under 1 to 3 years old, and 4 cases (14%) greater than or equal 3 years old. The onset time of 25 cases (86%) was from April to September. The diseases included 19 cases (66%) septicemia (2 of which were combined with suppurative meningitis), 10 cases (34%) non-sepsis group, including 7 cases bacteremia and 3 cases local suppurative infection (2 cases of osteomyelitis, 1 case of appendicitis with peritonitis). The clinical manifestations were fever in 29 cases (100%), diarrhea and abdominal pain in 18 cases (62%), cough and runny nose in 10 cases (34%). Eighteen cases (62%) were cured and 11 cases (38%) were improved by effective antibiotics treatment. C-reactive protein in sepsis group was significantly higher than that in non-sepsis group (25.2 (16.1, 56.4) vs. 3.4 (0.5, 7.5) mg/L, Z=-3.81, P<0.001).The serogroups of C, B and E were the most prevalent among non-typhoid Salmonella isolates, accounting for 10 cases (34%), 9 cases (31%) and 7 cases (24%) respectively. Antibacterial drug sensitivity test showed that the sensitivity rates of imipenem, ertapenem and piperaciratazobactam were all 100% (31/31), those of ceftazidime, ceftriaxone, and cefepime were 94% (29/31), 94% (29/31) and 97% (30/31) respectively. The drug resistance rates of ampicillin, ampicillin-sulbactam and trimethoprim-sulfamethoxazole were 51% (16/31), 48% (15/31) and 48% (15/31) respectively, those of cefazolin, cefotetan, tobramycin, gentamicin and amikacinwere all 100% (31/31). There were no significant differences in the drug resistance rates of ceftazidime, ceftriaxone, aztreonam, ampicillin-sulbactam, ampicillin, trimethoprim-sulfamethoxazole and ciprofloxacin between the sepsis group and the non-sepsis group (χ2=0.31,0.31,0.00,0.02,0.02,0.02,0.26, all P>0.05). Conclusions: Invasive non-typhoid Salmonella infection in children at Xiamen mainly occurred in infants younger than 3 years old.The main clinical manifestations are fever, abdominal pain and diarrhea. C-reactive protein can be served as the laboratory indicators for indicating sepsis. The third generation of cephalosporins is recommended as the first choice for treatment.

Fit to dwell in many places - The growing diversity of intracellular Salmonella niches.

Salmonella enterica is capable of invading different host cell types including epithelial cells and M cells during local infection, and immune cells and fibroblasts during the subsequent systemic spread. The intracellular lifestyles of Salmonella inside different cell types are remarkable for their distinct residential niches, and their varying replication rates. To study this, researchers have employed different cell models, such as various epithelial cells, immune cells, and fibroblasts. In epithelial cells, S. Typhimurium dwells within modified endolysosomes or gains access to the host cytoplasm. In the cytoplasm, the pathogen is exposed to the host autophagy machinery or poised for rapid multiplication, whereas it grows at a slower rate or remains dormant within the endomembrane-bound compartments. The swift bimodal lifestyle is not observed in fibroblasts and immune cells, and it emerges that these cells handle intracellular S. Typhimurium through different clearance machineries. Moreover, in these cell types S. Typhimurium grows withing modified phagosomes of distinct functional composition by adopting targeted molecular countermeasures. The preference for one or the other intracellular niche and the diverse cell type-specific Salmonella lifestyles are determined by the complex interactions between a myriad of bacterial effectors and host factors. It is important to understand how this communication is differentially regulated dependent on the host cell type and on the distinct intracellular growth rate. To support the efforts in deciphering Salmonella invasion across the different infection models, we provide a systematic comparison of the findings yielded from cell culture models. We also outline the future directions towards a better understanding of these differential Salmonella intracellular lifestyles.

Designed symmetrical ß-hairpin peptides for treating multidrug-resistant salmonella typhimurium infections.

The rapid emergence and prevalence of multidrug-resistant salmonellosis lack effective therapies, which causes epidemic health problems and stimulates the development of antimicrobials with novel modes of action. In this research, 10 short symmetrical ß-hairpin peptides are synthesized by combining the ß-turn of Leucocin-A with recurring hydrophobic and cationic amino acid sequences. Those designed peptides exhibited potent antibacterial activities against drug-susceptible and drug-resistant Salmonella. One of the 10 peptides, WK2 ((WK)2CTKSGC(KW)2), displayed best cell selectivity towards Salmonella cells over macrophages and erythrocytes in a co-culture model. Fluorescent measurements and microscopic observations reflected that WK2 exerted its antimicrobial activity through a membrane-lytic mechanism. Moreover, the ß-hairpin peptides can bind to endotoxin (LPS) and suppress the production of LPS-induced proinflammatory cytokines in RAW264.7 cells, indicating as a potent anti-inflammatory activity. The preliminary in vivo studies can also demonstrate that WK2 decreased loads of Salmonella in the liver and spleen, mitigated Salmonella-caused inflammation and maintained the integrity of intestinal mucosal surfaces. Ultimately, the results highlight that WK2 is a promising therapeutic agent to prevent multidrug-resistant S. Typhimurium infections in humans and animals.

Clinical profiles and antimicrobial resistance patterns of invasive Salmonella infections in children in China.

Invasive Salmonella infections result in a significant burden of disease including morbidity, mortality, and financial cost in many countries. Besides typhoid fever, the clinical impact of non-typhoid Salmonella infections is increasingly recognized with the improvement of laboratory detection capacity and techniques. A retrospective multicenter study was conducted to analyze the clinical profiles and antimicrobial resistance patterns of invasive Salmonella infections in hospitalized children in China during 2016-2018. A total of 130 children with invasive Salmonella infections were included with the median age of 12 months (range: 1-144 months). Seventy-nine percent of cases occurred between May and October. Pneumonia was the most common comorbidity in 33 (25.4%) patients. Meningitis and septic arthritis caused by nontyphoidal Salmonella (NTS) infections occurred in 12 (9.2%) patients and 5 (3.8%) patients. Patients < 12 months (OR: 16.04) and with septic shock (OR: 23.4), vomit (OR: 13.33), convulsion (OR: 15.86), C-reactive protein (CRP) ≥ 40 g/L (OR: 5.56), and a higher level of procalcitonin (PCT) (OR: 1.05) on admission were statistically associated to an increased risk of developing meningitis. Compared to 114 patients with NTS infections, 16 patients with typhoid fever presented with higher levels of CRP and PCT (P < 0.05). The rates of resistance to ampicillin, sulfamethoxazole/trimethoprim, ciprofloxacin, and ceftriaxone among Salmonella Typhi and NTS isolates were 50% vs 57.3%, 9.1% vs 24.8%, 0% vs 11.2%, and 0% vs 9.9%, respectively. NTS has been the major cause of invasive Salmonella infections in Chinese children and can result in severe diseases. Antimicrobial resistance among NTS was more common.

Cyclopropane Fatty Acids Are Important for Salmonella enterica Serovar Typhimurium Virulence.

A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the cfa gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study, we constructed a cfa mutant of Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and cfa mutant S. Typhimurium. CFA levels in the cfa mutant were greatly reduced compared to CFA levels in the wild type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. S. Typhimurium cfa mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide compared to the wild type. In addition, cfa mutants exhibited reduced viability in murine macrophages and could be rescued by the addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. S. Typhimurium lacking cfa was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.

Surface Glycans Regulate Salmonella Infection-Dependent Directional Switch in Macrophage Galvanotaxis Independent of NanH.

Salmonella invades and disrupts gut epithelium integrity, creating an infection-generated electric field that can drive directional migration of macrophages, a process called galvanotaxis. Phagocytosis of bacteria reverses the direction of macrophage galvanotaxis, implicating a bioelectrical mechanism to initiate life-threatening disseminations. The force that drives direction reversal of macrophage galvanotaxis is not understood. One hypothesis is that Salmonella can alter the electrical properties of the macrophages by modifying host cell surface glycan composition, which is supported by the fact that cleavage of surface-exposed sialic acids with a bacterial neuraminidase severely impairs macrophage galvanotaxis, as well as phagocytosis. Here, we utilize N-glycan profiling by nanoLC-chip QTOF mass cytometry to characterize the bacterial neuraminidase-associated compositional shift of the macrophage glycocalyx, which revealed a decrease in sialylated and an increase in fucosylated and high mannose structures. The Salmonella nanH gene, encoding a putative neuraminidase, is required for invasion and internalization in a human colonic epithelial cell infection model. To determine whether NanH is required for the Salmonella infection-dependent direction reversal, we constructed and characterized a nanH deletion mutant and found that NanH is partially required for Salmonella infection in primary murine macrophages. However, compared to wild type Salmonella, infection with the nanH mutant only marginally reduced the cathode-oriented macrophage galvonotaxis, without canceling direction reversal. Together, these findings strongly suggest that while neuraminidase-mediated N-glycan modification impaired both macrophage phagocytosis and galvanotaxis, yet to be defined mechanisms other than NanH may play a more important role in bioelectrical control of macrophage trafficking, which potentially triggers dissemination.

Antiinflammatory and antiinfective effect of caffeine in a mouse model of disseminated salmonellosis.

Caffeine has been reported for its antiinflammatory properties by stimulating phagocytosis. In this study, we investigated the antiinflammatory and antiinfective potential of caffeine in murine macrophage cell cultures and Swiss mice infected with virulent Salmonella enterica serotype typhimurium. Peritoneal macrophages (pMØ) were treated with caffeine on 96-well plates for 24 hr and then infected with Salmonella for 4 hr. In another experiment, the pMØ were first infected with the bacterium for 4 hr and then treated with caffeine for 24 hr. In addition, Swiss mice were inoculated, intraperitoneally, with S. typhimurium and then received caffeine intravenously. Control groups received phosphate-buffered saline (PBS) or dexamethasone. We found that treatments with caffeine increased the macrophage cell viability and reduced the intracellular bacterial load. The administration of caffeine to Swiss mice reduced the infiltration of leukocytes into the peritoneal cavity after the bacterial challenge. Furthermore, the bacterial burdens in the peritoneal fluid, bloodstream, spleen, and liver were decreased by caffeine treatment. The expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6, and inducible nitric oxide synthase (iNOs) were down-regulated after infection in caffeine-treated mice. We can conclude that caffeine has both antiinflammatory and antiinfective properties that can be useful for management of bacterial infections along with antibiotics.

A detrimental role of NLRP6 in host iron metabolism during Salmonella infection.

Maintaining host iron homeostasis is an essential component of nutritional immunity responsible for sequestrating iron from pathogens and controlling infection. Nucleotide-oligomerization domain-like receptors (NLRs) contribute to cytoplasmic sensing and antimicrobial response orchestration. However, it remains unknown whether and how NLRs may regulate host iron metabolism, an important component of nutritional immunity. Here, we demonstrated that NLRP6, a member of the NLR family, has an unconventional role in regulating host iron metabolism that perturbs host resistance to bacterial infection. NLRP6 deficiency is advantageous for maintaining cellular iron homeostasis in both macrophages and enterocytes through increasing the unique iron exporter ferroportin-mediated iron efflux in a nuclear factor erythroid-derived 2-related factor 2 (NRF2)-dependent manner. Additional studies uncovered a novel mechanism underlying NRF2 regulation and operating through NLRP6/AKT interaction and that causes a decrease in AKT phosphorylation, which in turn reduces NRF2 nuclear translocation. In the absence of NLRP6, increased AKT activation promotes NRF2/KEAP1 dissociation via increasing mTOR-mediated p62 phosphorylation and downregulates KEAP1 transcription by promoting FOXO3A phosphorylation. Together, our observations provide new insights into the mechanism of nutritional immunity by revealing a novel function of NLRP6 in regulating iron metabolism, and suggest NLRP6 as a therapeutic target for limiting bacterial iron acquisition.

Isolates of Salmonella typhimurium circumvent NLRP3 inflammasome recognition in macrophages during the chronic phase of infection.

Inflammasome signaling results in cell death and release of cytokines from the IL-1 family, which facilitates control over an infection. However, some pathogens such as Salmonella typhimurium (ST) activate various innate immune signaling pathways, including inflammasomes, yet evade these cell death mechanisms, resulting in a chronic infection. Here we investigated inflammasome signaling induced by acute and chronic isolates of ST obtained from different organs. We show that ST isolated from infected mice during the acute phase displays an increased potential to activate inflammasome signaling, which then undergoes a protracted decline during the chronic phase of infection. This decline in inflammasome signaling was associated with reduced expression of virulence factors, including flagella and the Salmonella pathogenicity island I genes. This reduction in cell death of macrophages induced by chronic isolates had the greatest impact on the NLRP3 inflammasome, which correlated with a reduction in caspase-1 activation. Furthermore, rapid cell death induced by Casp-1/11 by ST in macrophages limited the subsequent activation of cell death cascade proteins Casp-8, RipK1, RipK3, and MLKL to prevent the activation of alternative forms of cell death. We observed that the lack of the ability to induce cell death conferred a competitive fitness advantage to ST only during the acute phase of infection. Finally, we show that the chronic isolates displayed a significant attenuation in their ability to infect mice through the oral route. These results reveal that ST adapts during chronic infection by circumventing inflammasome recognition to promote the survival of both the host and the pathogen.

Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens.

Pathogenic bacteria can rapidly respond to stresses such as reactive oxygen species (ROS) using reversible redox-sensitive oxidation of cysteine thiol (-SH) groups in regulators. Here, we use proteomics to profile reversible ROS-induced thiol oxidation in Vibrio cholerae, the etiologic agent of cholera, and identify two modified cysteines in ArcA, a regulator of global carbon oxidation that is phosphorylated and activated under low oxygen. ROS abolishes ArcA phosphorylation but induces the formation of an intramolecular disulfide bond that promotes ArcA-ArcA interactions and sustains activity. ArcA cysteines are oxidized in cholera patient stools, and ArcA thiol oxidation drives in vitro ROS resistance, colonization of ROS-rich guts, and environmental survival. In other pathogens, such as Salmonella enterica, oxidation of conserved cysteines of ArcA orthologs also promotes ROS resistance, suggesting a common role for ROS-induced ArcA thiol oxidation in modulating ArcA activity, allowing for a balance of expression of stress- and pathogenesis-related genetic programs.

The interaction of enteric bacterial effectors with the host engulfment pathway control innate immune responses.

Host engulfment protein ELMO1 generates intestinal inflammation following internalization of enteric bacteria. In Shigella, bacterial effector IpgB1 interacts with ELMO1 and promotes bacterial invasion. IpgB1 belongs to the WxxxE effector family, a motif found in several effectors of enteric pathogens. Here, we have studied the role of WxxxE effectors, with emphasis on Salmonella SifA and whether it interacts with ELMO1 to regulate inflammation. In-silico-analysis of WxxxE effectors was performed using BLAST search and Clustal W program. The interaction of ELMO1 with SifA was assessed by GST pulldown assay and co-immunoprecipitation. ELMO1 knockout mice, and ELMO1-depleted murine macrophage J774 cell lines were challenged with WT and SifA mutant Salmonella. Bacterial effectors containing the WxxxE motif were transfected in WT and ELMO1-depleted J774 cells to assess the inflammatory cytokines. ELMO1 generates differential pro-inflammatory cytokines between pathogenic and nonpathogenic bacteria. WxxxE motif is present in pathogens and in the TIR domain of host proteins. The C-terminal part of ELMO1 interacts with SifA where WxxxE motif is important for interaction. ELMO1-SifA interaction affects bacterial colonization, dissemination, and inflammatory cytokines in vivo. Moreover, ELMO1-SifA interaction increases TNF-α and IL-6 production from the macrophage cell line and is associated with enhanced Rac1 activity. ELMO1 also interacts with WxxxE effectors IpgB1, IpgB2, and Map and induces inflammation after challenge with microbes or microbial ligands. ELMO1 generates a differential response through interaction with the WxxxE motif, which is absent in commensals. ELMO1-WxxxE interaction plays a role in bacterial pathogenesis and induction of inflammatory response.

Murine AML12 hepatocytes allow Salmonella Typhimurium T3SS1-independent invasion and intracellular fate.

Numerous studies have demonstrated the key role of the Salmonella Pathogenicity Island 1-encoded type III secretion system (T3SS1) apparatus as well as its associated effectors in the invasion and intracellular fate of Salmonella in the host cell. Several T3SS1 effectors work together to control cytoskeleton networks and induce massive membrane ruffles, allowing pathogen internalization. Salmonella resides in a vacuole whose maturation requires that the activity of T3SS1 subverts early stages of cell signaling. Recently, we identified five cell lines in which Salmonella Typhimurium enters without using its three known invasion factors: T3SS1, Rck and PagN. The present study investigated the intracellular fate of Salmonella Typhimurium in one of these models, the murine hepatocyte cell line AML12. We demonstrated that both wild-type Salmonella and T3SS1-invalidated Salmonella followed a common pathway leading to the formation of a Salmonella containing vacuole (SCV) without classical recruitment of Rho-GTPases. Maturation of the SCV continued through an acidified phase that led to Salmonella multiplication as well as the formation of a tubular network resembling Salmonella induced filaments (SIF). The fact that in the murine AML12 hepatocyte, the T3SS1 mutant induced an intracellular fate resembling to the wild-type strain highlights the fact that Salmonella Typhimurium invasion and intracellular survival can be completely independent of T3SS1.

B cell activating factor (BAFF) from neutrophils and dendritic cells is required for protective B cell responses against Salmonella typhimurium infection.

Mice lacking B cells are more susceptible to S. typhimurium infection. How B cells contribute to protective immunity against Salmonella and what signals drive their activation are still unclear. Neutrophils (Nphs), monocytes (MOs), and dendritic cells (DCs) are involved in early immune responses to control the initial replication of S. typhimurium. These cells can produce B cell activating factor (BAFF) required for mature B cell survival and may help regulate B cell responses during Salmonella infection. Using BAFF reporter mice (BAFF-RFP+/-), we discovered that an i.p. infection with a virulent strain of S. typhimurium increased BAFF expression in splenic conventional DCs (cDC) and inflammatory Ly6Chi MOs/DCs four days post-infection. S. typhimurium infection induced the release of BAFF from Nphs, a decrease of BAFF-RFP expression and expansion of BAFF-RFP+ Nphs in the spleen and peritoneal cavity. After S. typhimurium infection, serum BAFF levels and immature and mature B cell subsets and plasma cells increased substantially. Conditional knockout (cKO) mice lacking BAFF in either Nphs or cDCs compared to control Bafffl/fl mice had reduced up-regulation of systemic BAFF levels and reduced expansion of mature and germinal center B cell subsets after infection. Importantly, the cKO mice lacking BAFF from either Nphs or cDCs had impaired induction of Salmonella-specific IgM Abs, and were more susceptible to S. typhimurium infection. Thus, Nphs and cDCs are major cellular sources of BAFF driving B cell responses, required for mounting optimal protective immunity against lethal Salmonella infection.

Pyruvate Dehydrogenase Kinase Inhibitor Dichloroacetate Improves Host Control of Salmonella enterica Serovar Typhimurium Infection in Human Macrophages.

Global increases in the prevalence of antimicrobial resistance highlight the urgent need for novel strategies to combat infectious diseases. Recent studies suggest that host metabolic pathways play a key role in host control of intracellular bacterial pathogens. In this study we explored the potential of targeting host metabolic pathways for innovative host-directed therapy (HDT) against intracellular bacterial infections. Through gene expression profiling in human macrophages, pyruvate metabolism was identified as potential key pathway involved in Salmonella enterica serovar Typhimurium (Stm) infections. Next, the effect of targeting pyruvate dehydrogenase kinases (PDKs) - which are regulators of the metabolic checkpoint pyruvate dehydrogenase complex (PDC) - on macrophage function and bacterial control was studied. Chemical inhibition of PDKs by dichloroacetate (DCA) induced PDC activation and was accompanied with metabolic rewiring in classically activated macrophages (M1) but not in alternatively activated macrophages (M2), suggesting cell-type specific effects of dichloroacetate on host metabolism. Furthermore, DCA treatment had minor impact on cytokine and chemokine secretion on top of infection, but induced significant ROS production by M1 and M2. DCA markedly and rapidly reduced intracellular survival of Stm, but interestingly not Mycobacterium tuberculosis, in human macrophages in a host-directed manner. In conclusion, DCA represents a promising novel HDT compound targeting pyruvate metabolism for the treatment of Stm infections.

Macrophage Polarization Modulated by Porcine Circovirus Type 2 Facilitates Bacterial Coinfection.

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus-associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.

Balance between bacterial extracellular matrix production and intramacrophage proliferation by a Salmonella-specific SPI-2-encoded transcription factor.

Biosynthesis and secretion of a complex extracellular matrix (EM) is a hallmark of Salmonella biofilm formation, impacting on its relationship with both the environment and the host. Cellulose is a major component of Salmonella EM. It is considered an anti-virulence factor because it interferes with Salmonella proliferation inside macrophages and virulence in mice. Its synthesis is stimulated by CsgD, the master regulator of biofilm formation in enterobacteria, which in turn is under the control of MlrA, a MerR-like transcription factor. In this work, we identified a SPI-2-encoded Salmonella-specific transcription factor homolog to MlrA, MlrB, that represses transcription of its downstream gene, orf319, and of csgD inside host cells. MlrB is induced in laboratory media mimicking intracellular conditions and inside macrophages, and it is required for intramacrophage proliferation. An increased csgD expression is observed in the absence of MlrB inside host cells. Interestingly, inactivation of the CsgD-controlled cellulose synthase-coding gene restored intramacrophage proliferation to rates comparable to wild-type bacteria in the absence of MlrB. These data indicate that MlrB represses CsgD expression inside host cells and suggest that this repression lowers the activation of the cellulose synthase. Our findings provide a novel link between biofilm formation and Salmonella virulence.