Tellimagrandin-I and camptothin a exhibit chemopreventive effects in Salmonella enterica serovar Typhimurium strains and human lymphocytes.

Tellimagrandin-I (TL) and camptothin A (CA) are ellagitannins widely found in diverse plant species. Numerous studies demonstrated their significant biological activities, which include antitumor, antioxidant, and hepatoprotective properties. Despite this protective profile, the effects of TL and CA on DNA have not been comprehensively investigated. Thus, the aim of this study was to determine the mutagenic and antimutagenic effects attributed to TL and CA exposure on Salmonella enterica serovar Typhimurium strains using the Ames test. In addition, the cytotoxic and genotoxic effects were examined on human lymphocytes, employing both trypan blue exclusion and CometChip assay. The antigenotoxic effect was determined following TL and CA exposure in the presence of co-treatment with doxorubicin (DXR). Our results from the Ames test indicated that TL or CA did not display marked mutagenic activity. However, TL or CA demonstrated an ability to protect DNA against the damaging effects of the mutagens 4-nitroquinoline-1-oxide and sodium azide, thereby exhibiting antimutagenic properties. In relation to human lymphocytes, TL or CA did not induce significant cytotoxic or genotoxic actions on these cells. Further, these ellagitannins exhibited an ability to protect DNA from damage induced by DOX during co-treatment, indicating their potential beneficial usefulness as antigenotoxic agents. In conclusion, the protective effects of TL or CA against mutagens, coupled with their absence of genotoxic and cytotoxic effects on human lymphocytes, emphasize their potential therapeutic value in chemopreventive strategies.

Phosphorus starvation response and PhoB-independent utilization of organic phosphate sources by Salmonella enterica.

IMPORTANCE: Phosphorus (P) is the fifth most abundant element in living cells. This element is acquired mainly as inorganic phosphate (Pi, PO4 3-). In enteric bacteria, P starvation activates a two-component signal transduction system which is composed of the membrane sensor protein PhoR and its cognate transcription regulator PhoB. PhoB, in turn, promotes the transcription of genes that help maintain Pi homeostasis. Here, we characterize the P starvation response of the bacterium Salmonella enterica. We determine the PhoB-dependent and independent transcriptional changes promoted by P starvation and identify proteins enabling the utilization of a range of organic substrates as sole P sources. We show that transcription and activity of a subset of these proteins are independent of PhoB and Pi availability. These results establish that Salmonella enterica can maintain Pi homeostasis and repress PhoB/PhoR activation even when cells are grown in medium lacking Pi.

Salmonella enterica serovar Typhimurium genetic variants isolated after lethal treatment with Thymbra capitata essential oil (TCO) showed increased resistance to TCO in milk.

The high prevalence of Salmonella enterica in milk poses a risk of considerable concern in the preservation of certain dairy products, mainly those elaborated from raw milk. Essential oils (EOs) have been proposed as a promising food preservative for such products due to their strong antimicrobial properties. Additionally, these natural antimicrobials have been shown to be effective against multi-drug resistant strains. They can thus also be utilized to prevent the dissemination of antimicrobial resistances (AMR). However, recent evidence of the development of bacterial resistance under EO treatments may call their use into question. This study sought to assess the emergence of antimicrobial resistant genetic variants of S. enterica serovar Typhimurium from survivors after cyclic exposure to lethal doses (>5 log10 cycles of inactivation) of Thymbra capitata EO (TCO), in order to evaluate the impact that it could have on milk preservation, to ascertain whether cross-resistance to antibiotics occurs, and to identify the genomic changes responsible for their phenotype. Isolated strains by TCO (SeTCO) showed a two-fold increase in minimum inhibitory and bactericide concentrations (MIC and MBC) of TCO compared to Salmonella enterica serovar Typhimurium wild-type strain (SeWT) in laboratory growth medium, as well as a greater adaptation and growth rate in the presence of the EOs and a higher survival to TCO treatments in buffers of pH 4.0 and 7.0. The increased resistance of SeTCO was confirmed in skimmed milk: 300 µL/L TCO reduced only 1 log10 cycle of SeTCO population, whereas it inactivated more than 5 log10 cycles in SeWT. Moreover, SeTCO showed an increased cross-resistance against aminoglycosides, quinolones and tetracyclines. Whole genome sequencing revealed 5 mutations in SeTCO: 2 in genes involved in O-antigens synthesis (rfbV and rfbX), 2 in genes related to adaptation to the growing medium (trkA and glpK), and 1 in a redox-sensitive transcriptional regulator (soxR). The phenotypic characterization of a constructed SeWT strain with mutant soxRSeTCO demonstrated that the mutation of soxR was the main cause of the increased resistance and tolerance observed in SeTCO against TCO and antibiotics. The emergence of resistant strains against EOs might jeopardize their use as food preservatives. Further studies will thus be required to determine under which conditions such resistant strains might occur, and to assess the food risk they may pose, as well as to ascertain their impact on the spread of AMR.

A 3,000-year-old, basal S. enterica lineage from Bronze Age Xinjiang suggests spread along the Proto-Silk Road.

Salmonella enterica (S. enterica) has infected humans for a long time, but its evolutionary history and geographic spread across Eurasia is still poorly understood. Here, we screened for pathogen DNA in 14 ancient individuals from the Bronze Age Quanergou cemetery (XBQ), Xinjiang, China. In 6 individuals we detected S. enterica. We reconstructed S. enterica genomes from those individuals, which form a previously undetected phylogenetic branch basal to Paratyphi C, Typhisuis and Choleraesuis-the so-called Para C lineage. Based on pseudogene frequency, our analysis suggests that the ancient S. enterica strains were not host adapted. One genome, however, harbors the Salmonella pathogenicity island 7 (SPI-7), which is thought to be involved in (para)typhoid disease in humans. This offers first evidence that SPI-7 was acquired prior to the emergence of human-adapted Paratyphi C around 1,000 years ago. Altogether, our results show that Salmonella enterica infected humans in Eastern Eurasia at least 3,000 years ago, and provide the first ancient DNA evidence for the spread of a pathogen along the Proto-Silk Road.

An Unexpected Role for the Periplasmic Phosphatase PhoN in the Salvage of B6 Vitamers in Salmonella enterica.

Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B6, essential for cellular function in all domains of life. In many organisms, such as Salmonella enterica serovar Typhimurium and Escherichia coli, this cofactor can be synthesized de novo or salvaged from B6 vitamers in the environment. Unexpectedly, S. enterica strains blocked in PLP biosynthesis were able to use exogenous PLP and pyridoxine 5'-phosphate (PNP) as the source of this required cofactor, while E. coli strains of the same genotype could not. Transposon mutagenesis found that phoN was essential for the salvage of PLP and PNP under the conditions tested. phoN encodes a class A nonspecific acid phosphatase (EC 3.1.3.2) that is transcriptionally regulated by the PhoPQ two-component system. The periplasmic location of PhoN was essential for PLP and PNP salvage, and in vitro assays confirmed PhoN has phosphatase activity with PLP and PNP as substrates. The data suggest that PhoN dephosphorylates B6 vitamers, after which they enter the cytoplasm and are phosphorylated by kinases of the canonical PLP salvage pathway. The connection of phoN with PhoPQ and the broad specificity of the gene product suggest S. enterica is exploiting a moonlighting activity of PhoN for PLP salvage.IMPORTANCE Nutrient salvage is a strategy used by species across domains of life to conserve energy. Many organisms are unable to synthesize all required metabolites de novo and must rely exclusively on salvage. Others supplement de novo synthesis with the ability to salvage. This study identified an unexpected mechanism present in S. enterica that allows salvage of phosphorylated B6 vitamers. In vivo and in vitro data herein determined that the periplasmic phosphatase PhoN can facilitate the salvage of PLP and PNP. We suggest a mechanistic working model of PhoN-dependent utilization of PLP and PNP and discuss the general role of promiscuous phosphatases and kinases in organismal fitness.

Chromosomal and plasmid-mediated fluoroquinolone resistance in human Salmonella enterica infection in Ghana.

BACKGROUND: Salmonella infection poses significant public health threat globally, especially in resource-limited countries. Emergence and spread of antibiotic resistant strains to fluoroquinolones have led to treatment failures and increased mortality in Salmonella infection. However, there is dearth of information regarding mechanisms of resistance to fluoroquinolones in Ghana. This study therefore sought to identify chromosomal mutations and plasmid-mediated resistance as possible mechanisms of fluoroquinolone resistance from clinical isolates in Ghana. METHODS: This was a retrospective study of archived isolates biobanked at Kumasi Centre for Collaborative Research in Tropical Medicine, Ghana. Isolates were obtained from blood, stool and oropharynx samples at two hospitals, between May, 2016 and January, 2018. Salmonella identification was done using standard microbiological protocols and antibiotic susceptibility testing performed by Kirby-Bauer disc diffusion method. Isolates with intermediate susceptibility and/or resistance to nalidixic acid and/or ciprofloxacin were selected and examined for chromosomal mutations by Sanger sequencing and plasmid-mediated resistance by PCR. RESULTS: Of 133 biobanked isolates cultured, 68 (51.1%) and 16 (12%) were identified as Salmonella Typhi and non-typhoidal Salmonella (NTS), respectively. Sequence analysis of gyrA gene revealed the presence of 5 different nonsynonymous mutations, with the most frequent mutation (Ile203Ser) occurring in 12 out of 13 isolates tested. Gyrase B (gyrB) gene had 1 nonsynonymous mutation in 3 out of 13 isolates, substituting phenylalanine with leucine at codon 601 (Phe601Leu). No mutation was observed in parC and parE genes. Two NTS isolates were found to harbour qnrS plasmid-mediated resistant gene of molecular size 550 bp with high ciprofloxacin MIC of 0.5 µg/ml. CONCLUSION: This study reports for the first time in Ghana plasmid-mediated fluoroquinolone resistant gene qnrS in Salmonella clinical isolates. Nonsynonymous mutations of gyrA and gyrB genes likely to confer Salmonella reduced susceptibility to ciprofloxacin were also reported.

Genetic diversity and delineation of Salmonella Agona outbreak strains by next generation sequencing, Bavaria, Germany, 1993 to 2018.

BackgroundIn 2017, a food-borne Salmonella Agona outbreak caused by infant milk products from a French supplier occurred in Europe. Simultaneously, S. Agona was detected in animal feed samples in Bavaria.AimUsing next generation sequencing (NGS) and three data analysis methods, this study's objectives were to verify clonality of the Bavarian feed strains, rule out their connection to the outbreak, explore the genetic diversity of Bavarian S. Agona isolates from 1993 to 2018 and compare the analysis approaches employed, for practicality and ability to delineate outbreaks caused by the genetically monomorphic Agona serovar.MethodsIn this observational retrospective study, three 2017 Bavarian feed isolates were compared to a French outbreak isolate and 48 S. Agona isolates from our strain collections. The later included human, food, feed, veterinary and environmental isolates, of which 28 were epidemiologically outbreak related. All isolates were subjected to NGS and analysed by: (i) a publicly available species-specific core genome multilocus sequence typing (cgMLST) scheme, (ii) single nucleotide polymorphism phylogeny and (iii) an in-house serovar-specific cgMLST scheme. Using additional international S. Agona outbreak NGS data, the cluster resolution capacity of the two cgMLST schemes was assessed.ResultsWe could prove clonality of the feed isolates and exclude their relation to the French outbreak. All approaches confirmed former Bavarian epidemiological clusters.ConclusionEven for S. Agona, species-level cgMLST can produce reasonable resolution, being standardisable by public health laboratories. For single samples or homogeneous sample sets, higher resolution by serovar-specific cgMLST or SNP genotyping can facilitate outbreak investigations.

Enhanced squalene biosynthesis in Yarrowia lipolytica based on metabolically engineered acetyl-CoA metabolism.

As a bioactive triterpenoid, squalene is widely used in the food industry, cosmetics, and pharmacology. Squalene's major commercial sources are the liver oil of deep-sea sharks and plant oils. In this study, we focused on the enhancement of squalene biosynthesis in Yarrowia lipolytica, with particular attention to the engineering of acetyl-CoA metabolism based on genome-scale metabolic reaction network analysis. Although the overexpression of the rate-limiting endogenous ylHMG1 (3-hydroxy-3-methylglutaryl-CoA reductase gene) could improve squalene synthesis by 3.2-fold over that by the control strain, the availability of the key intracellular precursor, acetyl-CoA, was found to play a more significant role in elevating squalene production. Analysis of metabolic networks with the newly constructed genome-scale metabolic model of Y. lipolytica iYL_2.0 showed that the acetyl-CoA pool size could be increased by redirecting carbon flux of pyruvate dehydrogenation towards the ligation of acetate and CoA or the cleavage of citrate to form oxaloacetate and acetyl-CoA. The overexpression of either acetyl-CoA synthetase gene from Salmonella enterica (acs*) or the endogenous ATP citrate lyase gene (ylACL1) resulted in a more than 50% increase in the cytosolic acetyl-CoA level. Moreover, iterative chromosomal integration of the ylHMG1, asc*, and ylACL1 genes resulted in a significant improvement in squalene production (16.4-fold increase in squalene content over that in the control strain). We also found that supplementation with 10 mM citrate in a flask culture further enhanced squalene production to 10 mg/g DCW. The information obtained in this study demonstrates that rationally engineering acetyl-CoA metabolism to ensure the supply of this key metabolic precursor is an efficient strategy for the enhancement of squalene biosynthesis.

Antioxidant Activity and Genotoxic Assessment of Crabwood (Andiroba, Carapa guianensis Aublet) Seed Oils.

The seed oil of Carapa guianensis (Aublet), a tree from the Meliaceae family commonly known as andiroba, is widely used in Brazilian traditional medicine because of its multiple curative properties against fever and rheumatism and as an anti-inflammatory agent, antibacterial agent, and insect repellant. Since there is no consensus on the best way to obtain the C. guianensis oil and due to its ethnomedicinal properties, the aim of the present research was to evaluate the chemical composition, free-radical scavenging activity, and mutagenic and genotoxicity properties of three C. guianensis oils obtained by different extraction methods. The phenolic contents were evaluated by spectrophotometry. Oil 1 was obtained by pressing the dried seeds at room temperature; oil 2 was obtained by autoclaving, drying, and pressing; oil 3 was obtained by Soxhlet extraction at 30-60°C using petroleum ether. The oil from each process presented differential yields, physicochemical properties, and phenolic contents. Oil 1 showed a higher scavenging activity against the DPPH radical when compared to oils 2 and 3, suggesting a significant antioxidant activity. All oils were shown to be cytotoxic to bacteria and to CHO-K1 and RAW264.7 cells. At noncytotoxic concentrations, oil 2 presented mutagenicity to Salmonella enterica serovar Typhimurium and induced micronuclei in both cell types. Under the same conditions, oil 3 also induced micronucleus formation. However, the present data demonstrated that oil 1, extracted without using high temperatures, was the safest for use as compared to the other two oils, not showing mutagenicity or micronucleus induction.

A simple cell-based high throughput screening (HTS) assay for inhibitors of Salmonella enterica RNA polymerase containing the general stress response regulator RpoS (σS).

RNA polymerase containing the stress response regulator σS subunit (RpoS) plays a key role in bacterial survival in hostile environments in nature and during infection. Here we devise and validate a simple cell-based high throughput luminescence assay for this holoenzyme suitable for screening large chemical libraries in a robotic platform.

Measurement of Fructose-Asparagine Concentrations in Human and Animal Foods.

The food-borne bacterial pathogen, Salmonella enterica, can utilize fructose-asparagine (F-Asn) as its sole carbon and nitrogen source. F-Asn is the product of an Amadori rearrangement following the nonenzymatic condensation of glucose and asparagine. Heating converts F-Asn via complex Maillard reactions to a variety of molecules that contribute to the color, taste, and aroma of heated foods. Among these end derivatives is acrylamide, which is present in some foods, especially in fried potatoes. The F-Asn utilization pathway in Salmonella, specifically FraB, is a potential drug target because inhibition of this enzyme would lead to intoxication of Salmonella in the presence of F-Asn. However, F-Asn would need to be packaged with the FraB inhibitor or available in human foods. To determine if there are foods that have sufficient F-Asn, we measured F-Asn concentrations in a variety of human and animal foods. The 400 pmol/mg F-Asn found in mouse chow is sufficient to intoxicate a Salmonella fraB mutant in mouse models of salmonellosis, and several human foods were found to have F-Asn at this level or higher (fresh apricots, lettuce, asparagus, and canned peaches). Much higher concentrations (11 000-35 000 pmol/mg dry weight) were found in heat-dried apricots, apples, and asparagus. This report reveals possible origins of F-Asn as a nutrient source for Salmonella and identifies foods that could be used together with a FraB inhibitor as a therapeutic agent for Salmonella.

Deletion of nudB Causes Increased Susceptibility to Antifolates in Escherichia coli and Salmonella enterica.

Co-trimoxazole, a fixed-dose combination of sulfamethoxazole (SMX) and trimethoprim (TMP), has been used for the treatment of bacterial infections since the 1960s. Since it has long been assumed that the synergistic effects between SMX and TMP are the consequence of targeting 2 different enzymes of bacterial folate biosynthesis, 2 genes (pabB and nudB) involved in the folate biosynthesis of Escherichia coli were deleted, and their effects on the susceptibility to antifolates were tested. The results showed that the deletion of nudB resulted in a lag of growth in minimal medium and increased susceptibility to both SMX and TMP. Moreover, deletion of nudB also greatly enhanced the bactericidal effect of TMP. To elucidate the mechanism of how the deletion of nudB affects the bacterial growth and susceptibility to antifolates, 7,8-dihydroneopterin and 7,8-dihydropteroate were supplemented into the growth medium. Although those metabolites could restore bacterial growth, they had no effect on susceptibilities to the antifolates. Reverse mutants of the nudB deletion strain were isolated to further study the mechanism of how the deletion of nudB affects susceptibility to antifolates. Targeted sequencing and subsequent genetic studies revealed that the disruption of the tetrahydromonapterin biosynthesis pathway could reverse the phenotype caused by the nudB deletion. Meanwhile, overexpression of folM could also lead to increased susceptibility to both SMX and TMP. These data suggested that the deletion of nudB resulted in the excess production of tetrahydromonapterin, which then caused the increased susceptibility to antifolates. In addition, we found that the deletion of nudB also resulted in increased susceptibility to both SMX and TMP in Salmonella enterica Since dihydroneopterin triphosphate hydrolase is an important component of bacterial folate biosynthesis and the tetrahydromonapterin biosynthesis pathway also exists in a variety of bacteria, it will be interesting to design new compounds targeting dihydroneopterin triphosphate hydrolase, which may inhibit bacterial growth and simultaneously potentiate the antimicrobial activities of antifolates targeting other components of folate biosynthesis.

Heat resistance of Salmonella enterica is increased by pre-adaptation to peanut oil or sub-lethal heat exposure.

Cross-protection represents a considerable challenge in the food industry where hurdled interventions are often employed to reduce Salmonella contamination. The heat resistance of Salmonella strains from five serotypes (i.e., Typhimurium, Enteritidis, Tennessee, Thompson and Hartford) at 70 °C was determined by measurement of viable cell populations before and after adaptation to two common stresses employed in low-water activity food processing, desiccation and sub-lethal heat treatment. Survival of Salmonella at 70 °C significantly increased (p < 0.05) following the six-day incubation in peanut oil (aw 0.52 ± 0.00) and/or the exposure to a sub-lethal heat treatment at 45 °C for 3 min. Quantitative PCR revealed upregulation of two desiccation stress-related genes, fadA and otsB, following the peanut oil incubation, whereas heat treatment induced upregulation of a heat-resistance gene, dnaK. Invasion gene invA and alternative sigma factor rpoE were downregulated following either of the treatments. Interestingly, different Salmonella strains yielded different transcriptional profiles. The strain-specific resistance phenotypes and transcriptional profiles provided further insights into the mechanisms employed to tolerate desiccation and heat stresses in the food industry.

2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica.

UNLABELLED: The reactive enamine 2-aminoacrylate (2AA) is a metabolic stressor capable of damaging cellular components. Members of the broadly conserved Rid (RidA/YER057c/UK114) protein family mitigate 2AA stress in vivo by facilitating enamine and/or imine hydrolysis. Previous work showed that 2AA accumulation in ridA strains of Salmonella enterica led to the inactivation of multiple target enzymes, including serine hydroxymethyltransferase (GlyA). However, the specific cause of a ridA strain's inability to grow during periods of 2AA stress had yet to be determined. Work presented here shows that glycine supplementation suppressed all 2AA-dependent ridA strain growth defects described to date. Depending on the metabolic context, glycine appeared to suppress ridA strain growth defects by eliciting a GcvB small RNA-dependent regulatory response or by serving as a precursor to one-carbon units produced by the glycine cleavage complex (GCV). In either case, the data suggest that GlyA is the most physiologically sensitive target of 2AA inactivation in S. enterica. The universally conserved nature of GlyA among free-living organisms highlights the importance of RidA in mitigating 2AA stress. IMPORTANCE: The RidA stress response prevents 2-aminoacrylate (2AA) damage from occurring in prokaryotes and eukaryotes alike. 2AA inactivation of serine hydroxymethyltransferase (GlyA) from Salmonella enterica restricts glycine and one-carbon production, ultimately reducing fitness of the organism. The cooccurrence of genes encoding 2AA production enzymes and serine hydroxy-methyltransferase (SHMT) in many genomes may in part underlie the evolutionary selection for Rid proteins to maintain appropriate glycine and one-carbon metabolism throughout life.

Contribution of the Salmonella enterica KdgR Regulon to Persistence of the Pathogen in Vegetable Soft Rots.

During their colonization of plants, human enteric pathogens, such as Salmonella enterica, are known to benefit from interactions with phytopathogens. At least in part, benefits derived by Salmonella from the association with a soft rot caused by Pectobacterium carotovorum were shown to be dependent on Salmonella KdgR, a regulator of genes involved in the uptake and utilization of carbon sources derived from the degradation of plant polymers. A Salmonella kdgR mutant was more fit in soft rots but not in the lesions caused by Xanthomonas spp. and Pseudomonas spp. Bioinformatic, phenotypic, and gene expression analyses demonstrated that the KdgR regulon included genes involved in uptake and metabolism of molecules resulting from pectin degradation as well as those central to the utilization of a number of other carbon sources. Mutant analyses indicated that the Entner-Doudoroff pathway, in part controlled by KdgR, was critical for the persistence within soft rots and likely was responsible for the kdgR phenotype.

Development of ceftriaxone resistance in Salmonella enterica serotype Oranienburg during therapy for bacteremia.

BACKGROUND: The majority of nontyphoid Salmonella infection is identified in children. When an invasive or severe Salmonella infection is encountered, ceftriaxone is recommended for such patients. A 2-year-old girl was hospitalized for the treatment of Salmonella bacteremia and discharged with standard ceftriaxone treatment. She was readmitted to the hospital after 2 days due to the recurrence of the Salmonella bacteremia. The study aimed to unveil the mechanism for the relapse. METHODS: Six isolates (4 blood and 2 stool) were recovered from the patient, with the last two blood isolates being ceftriaxone-resistant. Pulsed-field gel electrophoresis was used for genotyping. Ceftriaxone resistance genes and transferability of the resistance plasmid were examined by molecular methods. RESULTS: All isolates were identified as Salmonella enterica serotype Oranienburg. Five isolates demonstrated almost identical electrophoresis patterns, except that in the two ceftriaxone-resistant isolates an extra band (>100 kb) was noted. A blaCMY-2 gene, carried by a 120-kb conjugative IncI1 plasmid of the sequence type 53, was identified in the two ceftriaxone-resistant isolates. Transfer of the resistance plasmid from one blood isolate to Escherichia coli J53 resulted in the increase of ceftriaxone minimum inhibitory concentration from 0.125 µg/mL to 32 µg/mL in the recipient. CONCLUSION: Ceftriaxone is the standard therapeutic choice for invasive or serious Salmonella infections in children. Pediatricians should be aware of the possibility of resistance development during therapy, especially in areas with a widespread of ceftriaxone resistance genes that are carried by a self-transferrable plasmid, such as the blaCMY-2-carrying IncI1 plasmid identified herein.

Role of host cell-derived amino acids in nutrition of intracellular Salmonella enterica.

The facultative intracellular pathogen Salmonella enterica resides in a specific membrane-bound compartment termed the Salmonella-containing vacuole (SCV). Despite being segregated from access to metabolites in the host cell cytosol, Salmonella is able to efficiently proliferate within the SCV. We set out to unravel the nutritional supply of Salmonella in the SCV with focus on amino acids. We studied the availability of amino acids by the generation of auxotrophic strains for alanine, asparagine, aspartate, glutamine, and proline in a macrophage cell line (RAW264.7) and an epithelial cell line (HeLa) and examined access to extracellular nutrients for nutrition. Auxotrophies for alanine, asparagine, or proline attenuated intracellular replication in HeLa cells, while aspartate, asparagine, or proline auxotrophies attenuated intracellular replication in RAW264.7 macrophages. The different patterns of intracellular attenuation of alanine- or aspartate-auxotrophic strains support distinct nutritional conditions in HeLa cells and RAW264.7 macrophages. Supplementation of medium with individual amino acids restored the intracellular replication of mutant strains auxotrophic for asparagine, proline, or glutamine. Similarly, a mutant strain deficient in succinate dehydrogenase was complemented by the extracellular addition of succinate. Complementation of the intracellular replication of auxotrophic Salmonella by external amino acids was possible if bacteria were proficient in the induction of Salmonella-induced filaments (SIFs) but failed in a SIF-deficient background. We propose that the ability of intracellular Salmonella to redirect host cell vesicular transport provides access of amino acids to auxotrophic strains and, more generally, is essential to continuously supply bacteria within the SCV with nutrients.

Phenotypic profile linked to inhibition of the major Zn influx system in Salmonella enterica: proteomics and ionomics investigations.

Zinc is required for a wide variety of cellular functions and plays a key role in bacterial metabolism and virulence. However, Zn can also be toxic and, therefore, its influx is tightly regulated. The high affinity zinc uptake transporter ZnuABC is the main Zn influx system in Salmonella enterica under conditions of Zn starvation. It has been shown that deletion of the gene encoding for its periplasmic subunit ZnuA significantly affects S. Typhimurium growth rate and virulence, highlighting the importance of this system in the host-pathogen interaction. To gain further insight into the mechanisms involved in Zn influx regulation, we characterized the main alterations in the ionome and proteome of S. Typhimurium wild type and znuA mutant strains grown either under Zn starvation or under Zn-replete conditions. We found significant differences in the element profile and protein expression that were reversed by Zn supplementation. In particular, several of the differentially regulated proteins are predicted to be metal-binding proteins. Interestingly, their over-expression in the znuA mutant strain strictly depends on Zn starvation and correlates with the differences found at the ionome level. In conclusion, our data demonstrate that inhibition of Zn influx has relevant effects either on the bacterial ionome or proteome and shed new light on the role of the ZnuABC system and Zn influx in S. Typhimurium pathogenicity.

[Construction of a promoter reporter array for antibiotic screening].

We designed and constructed an antibiotic screening system by using antibiotic responsive genes as reporters. Plasmid pCS26 carrying a promoterless luminescence reporter, luxCDABE, was used as the vector and the promoter regions of antibiotic responsive genes/operons from Escherichia coli were cloned upstream of the lux reporter to form the first part of the screening reporter array. Random promoter library of Salmonella enterica and Pseudomonas aeruginosa were screened for antibiotic responsive clones which consist of the second part of the screening array. The selected final reporter array responded to different antibiotics in distinct patterns and enabled in vivo high-throughput screening for antibiotics. Unknown antibiotics could, in general, be classified by analyzing the response patterns. This screening system is both sensitive and efficient and should prove to be a useful tool for screening new antibiotic compounds.

Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery.

The small regulatory protein Crl binds to sigmaS, the RNA polymerase stationary phase sigma factor. Crl facilitates the formation of the sigmaS-associated holoenzyme (EsigmaS) and thereby activates sigmaS-dependent genes. Using a real time surface plasmon resonance biosensor, we characterized in greater detail the specificity and mode of action of Crl. Crl specifically forms a 1:1 complex with sigmaS, which results in an increase of the association rate of sigmaS to core RNA polymerase without any effect on the dissociation rate of EsigmaS. Crl is also able to associate with preformed EsigmaS with a higher affinity than with sigmaS alone. Furthermore, even at saturating sigmaS concentrations, Crl significantly increases EsigmaS association with the katN promoter and the productive isomerization of the EsigmaS-katN complex, supporting a direct role of Crl in transcription initiation. Finally, we show that Crl does not bind to sigma70 itself but is able at high concentrations to form a weak and transient 1:1 complex with both core RNA polymerase and the sigma70-associated holoenzyme, leaving open the possibility that Crl might also exert a side regulatory role in the transcriptional activity of additional non-sigmaS holoenzymes.