Genetic Influences on Salmonella Formation

Genetic Influences on Salmonella Formation IHF Gene Influences Salmonella Enteritidis Biofilm Formation Integration Host Factor (IHF) is important for biofilm formation by Salmonella enterica Enteritidis Bruna Leite, Catierine Hirsch Werle, Camila Pinheiro do Carmo, Diego Borin Nbrega, Guilherme Paier Milanez, Cristina E. Alvarez-Martinez, Marcelo Brocchi Abstract Salmonella enterica Enteritidis forms biofilms and survives in agricultural environments where it infects poultry and eggs. Once established, biofilms are difficult to eradicate, due to their high resistance compared to planktonic cells, causing serious problems in industry and public health. In this study, we evaluated biofilm formation in wild-type strains of S. enterica Enteritidis and in ihf mutants employing different microbiology techniques. Our data indicate that ihf mutants display impaired biofilm formation, with a reduced of matrix formation and a decrease in CFU and metabolic activity. Phenotypic analysis indicated a deficiency in curli fimbriae expression and in cellulose production and pellicle formation. These results show that IHF has a regulatory role in biofilm formation in S. enterica Enteritidis. Keywords: Biofilm, Salmonella enterica Enteritidis, Polysaccharide matrix, Curli fimbriae, Cellulose, Integration Host Factor. Introduction A biofilm is defined as a bacterial colony adherent to a solid surface, which secretes a protective exopolysaccharide matrix. Every natural wet surface is a potential substrate for microbial biofilms. These sessile multicellular microbial consortia are embedded within self-produced extracellular polymeric substances (EPS). In food handling facilities, biofilms can be particularly problematic The ability to form biofilms is also an important factor in the virulence of S. Enterica. S. enterica subspecies I serovar Enteritidis is a leading cause of salmonellosis worldwide, and has emerged as one of the most important foodborne pathogens for humans. It is mainly associated with consumption of contaminated meat and eggs of poultry. A number of studies have demonstrated that S. enterica is capable of forming biofilms on a wide variety of contact surfaces, and the formation of biofilms may improve the ability of these organisms to resist stresses such as desiccation, extreme temperatures, antibiotics, and antiseptics. Biofilm formation allows S. enterica to survive for long periods in a poultry farm environment and to contaminate poultry meat and eggs, which remain the leading vehicles of salmonellosis outbreaks Many factors are involved in biofilm development. Curli fimbriae and cellulose are the major components of biofilm formed by S. enterica, whereas capsular polysaccharide, other polysaccharide-rich compounds such as lipopolyssaccharide (LPS), and a large secreted protein, BapA, also contribute to biofilm formation. Several regulatory genes involved in biofilm formation have been identified The expression of curli fimbriae and cellulose can be assayed phenotypically by growing enteric bacteria on Congo red indicator plates Bacteria may live in planktonic form in liquid media or as biofilms on biotic or abiotic surfaces. They need to adjust their genetic programs in order to switch from one lifestyle to another. The production of bacterial products and behaviours associated with environmental adaptation must be tightly coordinated to optimize the energy consumption. In bacteria, gene expression regulation is exerted primarily at the level of transcription initiation using a large array of transcription factors whose concentrations and activities change depending on specific environmental or metabolic signals. Topological changes in DNA also influence promoter recognition, open complex formation, and gene expression Nucleoid-associated proteins (NAPs) are global regulators of gene expression in bacteria. They alter the topology of DNA by bending, bridging, or wrapping it, leading to DNA transactions and multiple cellular effects that culminate in the modulation of gene expression. Integration-host factor (IHF) is a dimeric NAP that binds DNA in a sequence-specific manner and introduces curvatures of up to 180 °, which influence many aspects of bacterial physiology, including global gene expression, DNA topology, site-specific recombination, and DNA replication. In E. coli and S. enterica Typhimurium, the two IHF subunits-IHFÃŽ ± and IHFÃŽ ²-can assemble as hetero- or homo-dimers. There is also evidence indicating that the different dimeric forms of IHF regulate different but overlapping sets of genes Based on the global regulatory role of IHF, we hypothesized that this NAP can influence or directly regulate genes involved in biofilm formation in S. enterica Enteritidis. This hypothesis is supported by previous observations demonstrating that IHF activates curli production in S. enterica Typhimurium. Therefore, in this study, we evaluated the role of IHF genes in the initial stages of biofilm formation in S. Enteritidis. To this end, we performed phenotypic studies using isogenic deletion mutants of individual ihf genes (ihfA or ihfB) and a double mutant strain with deletions in both IHF subunits (ihfAB double mutant). Materials and methods Bacterial strains In this study, the S. enterica Enteritidis wild-type strain PT4SEn (IOC4647) provided a by the Fundaà §Ãƒ £o Oswaldo Cruz (FIOCRUZ, Rio de Janeiro, Brazil) was used. The draft genome of this strain was recently published (Milanez et al. 2016). It was found to be pathogenic in a mouse model assay (Carmo et al., unpublished results). The mutants of S. Enteritidis PT4SEn were previously constructed (Carmo et al., unpublished results) by deletion of ihf genes using the lambda Red system by transduction with P22HT phages. Mutant strains were designated as S. enterica Enteritidis PT4SEn ΔihfA, PT4SEn ΔihfB, and PT4SEn ΔihfAB. Bacterial growth conditions and storage Bacteria were cultivated in Luria-Bertani broth (LB) and on Luria-Bertani agar (LBA) plates prepared according to the method of Sambrook and Russell. All strains were stored at -80 °C in 30% glycerol All strains were inoculated from fresh LBA plates into 15 mL LB and grown for 18  ± 2 h at 37 °C in an orbital shaker at 140 rpm. Cells were harvested by centrifugation (for 5 min at 9,500 g and 4 °C) and resuspended in NaCl (0.9%) adjusted to 0.5 McFarland scale equivalent to 1.5 108 cells/mL prior to use in subsequent assays. Complementation of S. enterica Enteritidis ΔihfA and ΔihfB mutants Sequences corresponding to the ihfA and ihfB genes and their regulatory regions were obtained by PCR from the PT4SEn genome using the primers listed in Table 1. The DNA fragments were cloned in the pACYC184 vector (New England Biolabs, USA) between the NcoI and EcoRI restriction sites (restriction enzyme sites in the DNA fragments were introduced via the primers) and the vector was subsequently electroporated into the respective S. enterica Enteritidis mutant strains. Cloning, PCR amplification, electroporation, plasmid extraction, and agarose gel electrophoresis were performed as suggested by Sambrook and Russell (2001). After DNA purification using the Wizard ® Genomic DNA Purification Kit (Promega Corporation, Madison, USA), Sanger sequencing was performed using 3730XL Applied Biosystems (Foster City, California, USA) by the High Performance Technologies Central Laboratory in Life Sciences (LACTAD, University of Campinas UNICAMP, Campinas, Brazil). Biofilm formation on polystyrene plates Biofilms were formed in 96-well plates (Cell Culture Plate, Nest, Biotechnology Co, China) containing 200 ÃŽ ¼L of cell suspension (1 106 cells/mL) of S. enterica Enteritidis PT4SEn wild-type or mutant strains in LB supplemented with 0.25% of glucose. Plates were incubated at 37 °C with orbital shaking at 140 rpm for 48, 72, and 120 h. At the end of the incubation period, planktonic cells were carefully removed, and biofilms were washed twice with 200 ÃŽ ¼L of saline solution (0.9% NaCl). The crystal violet staining method was used to assess total biofilm biomass. Each well of the biofilm plates was incubated with 200 ÃŽ ¼L of methanol for 15 minutes. Subsequently, methanol was removed and 1% (v/v) crystal violet solution was added, followed by a 5-min incubation period. Wells were washed with distilled water and finally 33% (v/v) acetic acid was added. The absorbance was measured at 570 nm. The colorimetric method based on the reduction of XTT (2,3- bis(2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino)carbonyl-2H tetrazolium hydroxide; Sigma-Aldrich, USA) was used to determine cell activity (XTT is converted to a coloured formazan salt in the presence of metabolic activity). To each well of the biofilm plate, 200 ÃŽ ¼L of a solution containing 200 mg/L of XTT and 20 mg/L of phenazinemethosulphate (PMS; Sigma-Aldrich, Ukraine) was added. Microtiter plates were incubated for 3 h at 37 °C in the dark. The absorbance was measured at 490 nm. To assess the number of viable cells in biofilms, 200 ÃŽ ¼L of saline solution was added to each well before removal of the biofilm by scraping. For each sample, an aliquot of 1 mL (5 wells) was sonicated (20 s with 22% of amplitude; Ultrasonic Processor, Cole-Parmer, Illinois, USA) to promote biofilm disruption. The number of colony forming units (CFU) in biofilms was determined by performing 10-fold serial dilutions in saline solution, plating on LBA plates in triplicate, and incubating for 24 h. Scanning electron microscopy (SEM) of biofilm cells Biofilms of S. enterica Enteritidis PT4SEn wild-type and mutant strains formed in 24-well plates (Well Cell Culture Cluster, Costar) were dehydrated by a 15-min immersion in increasing ethanol concentrations (70, 95, and 100% ethanol [v/v]) and placed in sealed desiccators. The samples were mounted on aluminium stubs with carbon tape, sputter-coated with gold, and analysed with a JEOL JSM-5800LV scanning microscope. All experiments were carried out in duplicate. Biofilm formation at the air-liquid interface Biofilm formation at the air-liquid interface was assessed in S. enterica Enteritidis PT4SEn strains by inoculation of LB cultures without NaCl, followed by incubation at 28 °C without shaking. Every day for 10 days, each isolate was visually examined for pellicle formation. Experiments were performed in triplicate. Expression of curli fimbriae Bacterial colony morphology of S. enterica Enteritidis PT4SEn wild-type and mutant strains was analysed on LB agar without NaCl, supplemented with Congo red (1.01340.0025, Sigma-Aldrich, Germany; 40 ÃŽ ¼g/mL) and Coomassie brilliant blue G (B0770-5G, Sigma-Aldrich, China; 20 ÃŽ ¼g/mL). Bacterial cultures were spread on agar plates and the colour and degree of colony rugosity were determined after 96 h of growth at 28 °C. Images were captured with a camera (Nikon P500) and under an HBO 100 Carl Zeiss Illuminating microscope system. Cellulose production The fluorescence exhibited by bacteria after growth of S. enterica Enteritidis PT4SEn wild-type and mutant strains in LB plates with Calcofluor (Fluorescent Brightener 28; F3543-1G, Sigma-Aldrich, China; 200 ÃŽ ¼g/mL) served as an indicator of cellulose production. Fluorescence was analysed visually using an UV light (366 nm) after 48 h of growth at 37 °C. Statistical analysis Data were analysed using STATA software, version 13.0 (Stata Corp, College Station, TX, USA). Data from all assays were compared using one-way analysis of variance (ANOVA). Sidaks adjustment for multiple comparisons was performed after a significant fitting. The significance level was set at 5%. Results ihf mutants display reduced viability, biomass, and metabolic activity A decrease of about 1-2 log10 in number of viable cells was observed for the ihf mutants in comparison with the wild-type S. enterica Enteritidis PT4SEn strain by CFU counting (Figure 1-A). The differences observed were statistically significant (P < 0.05) for all periods of time evaluated. The introduction of the pACYC184 plasmid carrying ihfA or ihfB was generally associated with an increase in CFUs, but complementation did not completely restore the values to those obtained with the wild-type strain. No statistical differences were observed at 48 and 72 h of incubation between ΔihfAc and the wild-type strain. The same observation is valid for ΔihfB after 120 h of incubation (Figure 1-A). These results show that the restoration of ihfA or ihfB gene copies in mutant strains is generally associated with an increase in CFUs in biofilms. The total biofilm biomass, assessed by CV staining of S. enterica Enteritidis PT4SEn and mutant strains is presented in Figure 1-B. An increase in biomass is observed for the wild-type strain over time. However, this effect was not observed for the correspondent PT4SEn ihfAB double mutant. None of the mutants presented an increase in biofilm matrix density at 48 and 72 h of incubation (P < 0.05). The complemented PT4SEn ihfA and ihfB mutants (ihfAc and ihfBc) showed an increase in total biofilm biomass in comparison to the non-complemented mutants (Figure 1-B). All mutant strains exhibited a significant reduction in metabolic activity measured by the XTT assay for cells in biofilm (P < 0.05). In addition, the double mutant (ihfAB) showed the greatest reduction in metabolic activity at 72 and 120 h (Figure 1-C). ihf genes are essential for biofilm structure To further characterize biofilm formation and structure in strains lacking ihf genes, we performed scanning electron microscopy (SEM) analysis of cells in biofilms. As shown in Figure 2, the absence of ihfA or ihfB drastically affects biofilm formation, as null mutants of S. enterica Enteritidis PT4SEn (Figure 2-D, E and F) exhibited a low amount of matrix and small number of cells compared to the wild-type (Figure 2-A). Complementation of ihf gene deletions by a wild-type copy of the corresponding gene promoted a significant restoration of biofilm formation (Figure 2-B and C). Pellicle formation at the air-liquid interface To further characterize the mutant strains with respect to their ability to form biofilms we analysed the biofilm formation at the air-liquid interface of cultures of the different strains. Cultures of the wild-type strain formed a thick and rigid pellicle after 10 days of growth (Figure 3-A). On the other hand, PT4SEn ihfA or PT4SEn ihfB mutant strains formed a less compact and fragile pellicle (not shown). Interestingly, the double mutant strain PT4SEn ihfAB did not form a visible pellicle at all at the air-liquid interface. Instead, cell deposition was observed at the bottom of the tube (Figure 3-B). Complementation with the wild-type copy of ihfA and ihfB restored the phenotype of the single mutants (PT4SEn ΔihfAc and PT4SEn ΔihfBc strains), which now formed a thick and rigid pellicle (not shown). Curli and cellulose Since curli and cellulose are important components in biofilm formation, we evaluated the role of IHF on their production. To this end, colony morphology was analysed on LBA plates supplemented with Congo red and Coomassie brilliant blue, as previously described.. enterica Enteritidis PT4SEn wild-type and PT4SEn ΔihfA and ΔihfB complemented strains exhibited a phenotype consistent with curli fimbriae and cellulose production, with red, dry, and rough (rdar) colony morphology (Figure 4-A to D). However, the PT4SEn ΔihfA, PT4SEn ΔihfB, and PT4SEn ΔihfAB mutants of S. enterica Enteritidis did not display the same colour and roughness, but instead exhibited a similar, but not identical, smooth and white (saw) morphotype, indicating a deficiency in the expression of curli fimbriae and probably also of cellulose (Figure 4-E to H). The expression of cellulose was also tested by screening the colonies for Calcofluor binding Cellulose production was observed for all strain s evaluated by this method, except for the double mutant ihfAB that was not fluorescent under an UV light source and was considered a poor producer of cellulose (Figure 5). Discussion The presence of microorganisms on food contact surfaces is one of the most common causes of food spoilage and transmission of foodborne diseases. Inadequate cleaning and disinfection of food-processing environments is the cause of major economic losses and represents a serious danger to public health. The ability of microorganisms to adhere and form biofilms makes disinfection even more difficult and challenging Infections with Salmonella enterica Enteritidis represent a major health problem and a significant burden on the food industry. About 80% of the infections are caused by biofilm formation In the matrix of a biofilm, bacteria grow on either biotic or abiotic surfaces, attaching to the surface and to each other, conferring resistance to immunity responses as well as antimicrobial agents As a consequence, antimicrobial treatments typically fail to eradicate biofilms. The need to create effective therapies to counteract biofilm infections is a pressing challenge in the food indus try The growing interest in understanding the regulatory network of gene activities during the transition from a planktonic to a sessile cellular lifestyle, prompted us to investigate the role of IHF in S. enterica Enteritidis biofilm formation. IHF has an important role in the regulation of gene expression and environment adaptability of S. Enterica Therefore, S. Enteritidis deletion mutants for ihfA, ihfB, or both genes (ihfAB) were employed in different assays to analyse biofilm formation. The logic behind this approach is based on the fact that IHF can act as a homodimer (IHFÃŽ ±ÃŽ ± or IHFÃŽ ²ÃŽ ²) or as a heterodimer (IHFÃŽ ±ÃŽ ²) The results presented here indicate an important role of this NAP in the formation of biofilms in S. enterica Enteritidis. All typical biofilm characteristics analysed in this study (CFU, biomass, and cellular metabolic activity) were significantly decreased in S. enterica Enteritidis mutant strains for ihfA, ihfB, or ihfAihfB. The biofilms formed by mutant strains exhibited a decreased matrix density compared with the wild-type strain. Therefore, these results indicate that IHF can influence the initial stage of biofilm formation by S. enterica Enteritidis, as the matrix is necessary in this phase. This is also supported by CV staining and SEM. The colony morphotypes observed in Congo red among wild-type and complemented strains exhibited the rdar morphotype, an indication of curli and cellulose production, while the mutant strains exhibited a similar but not identical saw morphotype, suggesting an altered expression of curli and probably also of cellulose. In fact, bacterial growth in calcofluor-containing medium indicated that the single ihf-mutants were able to produce cellulose, but the ihf-double mutant exhibited some deficiency in the production of this polysaccharide. Previously, Gerstel, Park, and Rà ¶mling demonstrated that the ΔihfAB double mutant of two S. enterica Typhimurium strains caused a reduction in CsgD expression and an altered rdar morphotype suggesting a role for IHF in curli expression in S. enterica Typhimurium. Curli is expressed by two divergent operons, csgBAC and csgDEFG. CsgD is a major regulator of curli expression and biofilm formation. This gene activates transcription of csgA and csgB that encodes the major (CsgA) and the minor (CsgB) curli subunits In addition, csgD also regulates cellulose production Therefore, IHF plays an important role in biofilm formation in S. enterica Typhimurium. Our results demonstrate a similar role for IHF in the biofilm formation of S. enterica Enteritidis. Despite high genetic similarity, the Enteritidis and Typhimurium serovars differ in various ecological and host-relationship parameters However, the regulation of biofilm formation by IHF in both serovars suggests that IHF plays a cen tral role in S. enterica biofilm biogenesis. However, additional studies of IHF function on biofilm biogenesis in other S. enterica serovars are needed to further clarify this question. In addition, the single ihf mutants also exhibited a phenotypic alteration in biofilm formation, indicating that both subunits are necessary for appropriate biofilm production. In our results, all the ihf mutants showed a deficiency for curli fimbriae production by phenotypic tests. To some extent, a deficiency in cellulose production was also observed, particularly in the double ihf-mutant. The complementation of the ihfA and ihfB mutants by the introduction of a pACYC184 plasmid carrying the wild-type genes reverted the deficiency in biofilm biomass, cell metabolism, and CFUs, but in the majority of the tests the values did not reach those observed for the wild-type strain. This is probably due to a dose effect of IHFÃŽ ± or IHFÃŽ ², despite the low copy number (about 15 copies per cell) of the plasmid used. In fact, the expression of ihf genes is finely regulated and depends on the growth phase The two operons bcsABZC and bcsEFG are responsible for cellulose biosynthesis in both S. enterica Enteritidis and S. enterica Typhimurium. This was demonstrated by the construction of non-polar mutants of bcsC and bcsE genes that formed a fragile pellicle at the air-liquid interface of LB medium The same authors also showed that cellulose-deficient mutants were more sensitive to chlorine treatments, indicating that the deficiency in the production of extracellular matrix can leave the cells more susceptible to the action of some chemical agents. In our study, IHF mutant strains formed a less compact pellicle in LB compared to wild-type strains. In addition, the ihf double mutant did not form an air pellicle at all, suggesting a role for IHF in the expression of cellulose. These findings corroborate a previous study in which S. enterica Typhimurium ihfAB mutants exhibited reduced bcsC transcription when evaluated by microarray analysis, but further studies are needed to better charact erize the underlying molecular mechanisms. Karaca, N Akcelik, and M Akcelik (2013) also evaluated pellicle formation at the air-liquid interface of 31 S. enterica isolates. They showed that the growth rate of isolates with a rigid pellicle was greater than that of the ones forming a fragile pellicle. Biofilm production at the air-liquid interface can facilitate and contribute to gas exchange, while enabling the acquisition of nutrients and water from the liquid phase. Biofilms at air-liquid and solid-air interfaces can cause serious problems in industrial water systems. In conclusion, our results indicate that IHF has an important regulatory role in biofilm formation of S. enterica serovar Enteritidis. Moreover, both IHF subunits appear to have a role in this process. Our data pave the way for further studies investigating the mechanisms involved in the regulation of biofilm formation by IHF. Acknowledgements This work was supported by grants from Fundaà §Ãƒ £o de Amparo à   Pesquisa do Estado de Sà £o Paulo (FAPESP 2014/13412-8) and Conselho Nacional de Desenvolvimento Cientà ­fico e Tecnolà ³gico (CNPq), Brazil. BL, DBN, and GPM were supported by a FAPESP fellowship (FAPESP 2012/25426-8, 2012/10608-3, and 2012/05382-6, respectively). CHW and CPC were supported by fellowships from CNPq (141629/2012-6 and 140786/2012-0, respectively). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest or conflict.