Genome-wide characterization of Salmonella Typhimurium genes required for the fitness under iron restriction.

BACKGROUND: Iron is a crucial element for bacterial survival and virulence. During Salmonella infection, the host utilizes a variety of mechanisms to starve the pathogen from iron. However, Salmonella activates distinctive defense mechanisms to acquire iron and survive in iron-restricted host environments. Yet, the comprehensive set of the conditionally essential genes that underpin Salmonella survival under iron-restricted niches has not been fully explored. RESULTS: Here, we employed transposon sequencing (Tn-seq) method for high-resolution elucidation of the genes in Salmonella Typhimurium (S. Typhimurium) 14028S strain required for the growth under the in vitro conditions with four different levels of iron restriction achieved by iron chelator 2,2'-dipyridyl (Dip): mild (100 and 150 µM), moderate (250 µM) and severe iron restriction (400 µM). We found that the fitness of the mutants reduced significantly for 28 genes, suggesting the importance of these genes for the growth under iron restriction. These genes include sufABCDSE, iron transport fepD, siderophore tonB, sigma factor E ropE, phosphate transport pstAB, and zinc exporter zntA. The siderophore gene tonB was required in mild and moderate iron-restricted conditions, but it became dispensable in severe iron-restricted conditions. Remarkably, rpoE was required in moderate and severe iron restrictions, leading to complete attenuation of the mutant under these conditions. We also identified 30 genes for which the deletion of the genes resulted in increased fitness under iron-restricted conditions. CONCLUSIONS: The findings broaden our knowledge of how S. Typhimurium survives in iron-deficient environments, which could be utilized for the development of new therapeutic strategies targeting the pathways vital for iron metabolism, trafficking, and scavenging.

Genome-Wide Identification of Pseudomonas aeruginosa Genes Important for Desiccation Tolerance on Inanimate Surfaces.

Pseudomonas aeruginosa is an opportunistic pathogen prevalent in the environment and in health care settings. Transmission in the health care setting occurs through human-human interactions and/or contact with contaminated surfaces. Moist surfaces such as respirators, sink and tub drains, and even disinfectants can serve as reservoirs. Dry surfaces such as plastic and stainless steel could also serve as a reservoir but would necessitate some degree of tolerance to desiccation. Using an assay to measure P. aeruginosa tolerance to desiccation on plastic and stainless-steel surfaces, we found that only 0.05 to 0.1% of the desiccated cells could be recovered 24 h postdesiccation. We took advantage of the strong selection imposed by desiccation to identify genes important for tolerance using Tn-seq. A highly saturated Tn-seq library was desiccated on plastic and stainless-steel surfaces. NexGen sequencing of the recovered cells identified 97 genes important for survival. Comparing cells desiccated under low- and high-nutrient conditions allowed for differentiation of genes important for desiccation tolerance. The 53 genes identified in the latter analysis are involved in maintenance of cell envelope integrity, purine and pyrimidine biosynthesis, tricarboxylic acid (TCA) cycle, and the hydrolysis of misfolded proteins. The Tn-seq findings were validated by competition experiments with wild-type (WT) cells and select Tn insertion mutants. Mutants lacking carB and surA demonstrated the largest fitness defects, indicating that pyrimidine biosynthesis and outer membrane integrity are essential for desiccation tolerance. Increased understanding of desiccation tolerance could provide insight into approaches to control environmental reservoirs of P. aeruginosa. IMPORTANCE Health care-associated infections (HAIs) caused by Pseudomonas aeruginosa result in significant morbidity and mortality and are a significant economic burden. Moist environments that promote biofilm formation are an important reservoir for P. aeruginosa. Dry environments may also serve as a reservoir but would require some degree of desiccation tolerance. Here, we took a genome-wide approach to identify genes important for desiccation tolerance on plastic and stainless-steel surfaces. Genes involved in assembly of outer membrane proteins and pyrimidine biosynthesis were particularly important. Strains lacking these functions were unable to tolerate surface desiccation. These findings suggest that inhibitors of these pathways could be used to prevent P. aeruginosa survival on dry surfaces.

Genome Sequences of Two Pseudomonas aeruginosa Isolates with Defects in Type III Secretion System Gene Expression from a Chronic Ankle Wound Infection.

Effector proteins translocated into host cells by the Pseudomonas aeruginosa type III secretion system (T3SS) are critical for phagocytic avoidance and systemic spread of the microorganism. The T3SS genes are present in virtually all P. aeruginosa strains. When examined in environmental isolates and clinical specimens, expression of the T3SS genes is the rule. Isolates from the airways of cystic fibrosis (CF) patients are one exception, and these isolates usually carry mutations that disable T3SS gene expression. In this study, we describe two P. aeruginosa isolates, one pigmented brown and one green, from a keratitis-ichthyosis-deafness (KID) syndrome patient with a chronic cutaneous ankle wound. Similar to most isolates from CF, both of the KID isolates were defective for T3SS gene expression. Providing the primary activator of T3SS transcription (exsA) in trans restored T3SS function. Since the exsA sequences were identical to that of a reference strain with active T3SS gene expression, we examined the cAMP-Vfr system, a critical regulator of T3SS gene expression. Vfr is a cAMP-dependent transcription factor that activates exsA expression. Whereas T3SS activity was corrected in the brown isolate by restoring cAMP synthesis, the same was not observed for the green isolate. These findings suggest that distinct mechanisms resulted in loss of T3SS gene expression in the KID isolates. The mutations responsible for the T3SS defects were not clearly evident by comparison of the whole-genome sequences to a reference strain. Our findings suggest that loss of T3SS gene expression may be a trait common to both CF and non-CF chronic infections. IMPORTANCE A common feature of microorganisms that cause chronic infections is a stealthy lifestyle that promotes immune avoidance and host tolerance. During chronic colonization of cystic fibrosis (CF) patients, Pseudomonas aeruginosa acquires numerous adaptations that include reduced expression of some factors, such as motility, O antigen, and the T3SS, and increased expression of other traits, such as biofilm formation. In this study, we report loss of T3SS gene expression in non-CF chronic isolates. This finding suggests that loss of the T3SS may be a common and important trait that contributes to persistence and may open avenues to explore the significance further using non-CF chronic infection models.

Cautionary Notes on the Use of Arabinose- and Rhamnose-Inducible Expression Vectors in Pseudomonas aeruginosa.

The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of vfr transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed vfr transcription under the control of the rhaBp rhamnose-inducible promoter system (designated PRha) and found that PRha promoter activity was highly dependent upon vfr. Vfr dependence was also observed for the araBp arabinose-inducible promoter (designated PBAD). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the PRha and PBAD promoters in vitro. Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in PRha and PBAD promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution. IMPORTANCE Heterologous gene expression and complementation constitute a valuable and widely utilized tool in bacterial genetics. The arabinose-inducible ParaBAD (PBAD) and rhamnose-inducible PrhaBAD (PRha) promoter systems are commonly used in P. aeruginosa genetics and prized for the tight control and dynamic expression ranges that can be achieved. In this study, we demonstrate that the activity of both promoters is dependent upon the cAMP-dependent transcription factor Vfr. While this poses an obvious problem for use in a vfr mutant background, the issue is more pervasive, considering that vfr transcription/synthesis and cAMP homeostasis are highly integrated into the cellular physiology of the organism and influenced by numerous global regulatory systems. Fortunately, the synthetic PTac promoter is not subject to Vfr regulatory control.

Preparation of Transposon Library and Tn-Seq Amplicon Library for Salmonella Typhimurium.

Transposon sequencing (Tn-seq) is a powerful tool for functional genomics of bacteria. Tn-seq combines transposon mutagenesis with next generation sequencing to assess genetic requirements at a genome-wide scale and identify essential and conditionally essential genes. An efficient application of this experimental approach relies on robust protocols for transposon mutagenesis system and Tn-seq amplicon library preparation method. However, the existing approaches for the Tn-seq amplicon library preparation have several shortcomings. Hence, we present a robust, fast, specific, and cost-effective approach for the transposon mutagenesis of Salmonella Typhimurium and Tn-seq amplicon library preparation for Illumina sequencing. Besides S. Typhimurium that was used here for illustration, this protocol can also be used for other bacteria. In particular, the procedure for Tn-seq amplicon library preparation can be broadly applicable to any transposon elements. We delineate comprehensive step-by-step protocols for transposon mutagenesis and Tn-seq amplicon library such that it can be reproduced effortlessly by other researchers.

Iron-dependent essential genes in Salmonella Typhimurium.

BACKGROUND: The molecular mechanisms underlying bacterial cell death due to stresses or bactericidal antibiotics are complex and remain puzzling. Due to the current crisis of antibiotic resistance, development of effective antibiotics is urgently required. Previously, it has been shown that iron is required for effective killing of bacterial cells by numerous bactericidal antibiotics. RESULTS: We investigated the death or growth inhibition of S. Typhimurium under iron-restricted conditions, following disruption of essential genes, by transposon mutagenesis using transposon sequencing (Tn-seq). Our high-resolution Tn-seq analysis revealed that transposon mutants of S. Typhimurium with insertions in essential genes escaped immediate killing or growth inhibition under iron-restricted conditions for approximately one-third of all previously known essential genes. Based on this result, we classified all essential genes into two categories, iron-dependent essential genes, for which the insertion mutants can grow slowly if iron is restricted, and iron-independent essential genes, for which the mutants become nonviable regardless of iron concentration. The iron-dependency of the iron-dependent essential genes was further validated by the fact that the relative abundance of these essential gene mutants increased further with more severe iron restrictions. Our unexpected observation can be explained well by the common killing mechanisms of bactericidal antibiotics via production of reactive oxygen species (ROS). In this model, iron restriction would inhibit production of ROS, leading to reduced killing activity following blocking of essential gene functions. Interestingly, the targets of most antibiotics currently in use clinically are iron-dependent essential genes. CONCLUSIONS: Our result suggests that targeting iron-independent essential genes may be a better strategy for future antibiotic development, because blocking their essential gene functions would lead to immediate cell death regardless of the iron concentration. This work expands our knowledge on the role of iron to a broad range of essential functions and pathways, providing novel insights for development of more effective antibiotics.

A Comprehensive Assessment of the Genetic Determinants in Salmonella Typhimurium for Resistance to Hydrogen Peroxide Using Proteogenomics.

Salmonella is an intracellular pathogen infecting a wide range of hosts and can survive in macrophages. An essential mechanism used by macrophages to eradicate Salmonella is production of reactive oxygen species. Here, we used proteogenomics to determine the candidate genes and proteins that have a role in resistance of S. Typhimurium to H2O2. For Tn-seq, a saturated Tn5 insertion library was grown in vitro under either 2.5 (H2O2L) or 3.5 mM H2O2 (H2O2H). We identified two sets of overlapping genes required for resistance of S. Typhimurium to H2O2L and H2O2H, and the results were validated via phenotypic evaluation of 50 selected mutants. The enriched pathways for H2O2 resistance included DNA repair, aromatic amino acid biosynthesis (aroBK), Fe-S cluster biosynthesis, iron homeostasis and a putative iron transporter system (ybbKLM), and H2O2 scavenging enzymes. Proteomics revealed that the majority of essential proteins, including ribosomal proteins, were downregulated upon exposure to H2O2. On the contrary, a subset of conditionally essential proteins identified by Tn-seq were analyzed by targeted proteomics, and 70% of them were upregulated by H2O2. The identified genes will deepen our understanding on S. Typhimurium survival mechanisms in macrophages, and can be exploited to develop new antimicrobial drugs.

Rapid detection of avian influenza virus H5N1 in chicken tracheal samples using an impedance aptasensor with gold nanoparticles for signal amplification.

Highly pathogenic avian influenza virus H5N1 is a continuous threat to public health and poultry industry. The recurrence of the H5N1 led us to develop a robust, specific, and rapid detection method for the virus. In this study, an impedance aptasensor was developed for the virus detection using specific H5N1 aptamer and a gold interdigitated microelectrode. Streptavidin was immobilized on the microelectrode surface and biotin labeled H5N1 aptamer was bound to the immobilized streptavidin. The microelectrode was blocked with the polyethylene glycol and the bound aptamer captured the virus. The impedance change caused by the captured virus was measured using an impedance analyzer. To enhance impedance signal, a nanoparticle-based amplifier was designed and implemented by forming a network-like gold nanoparticles/H5N1-aptamer/thiocyanuric acid. The detection limit of the impedance aptasensor was 0.25 HAU for the pure virus and 1 HAU for the tracheal chicken swab samples spiked with the H5N1 virus. The detection time of aptasensor without employing the amplifier was less than an hour. The amplifier increased impedance by a 57-fold for the 1 HAU samples. Only negligible impedance change was observed for non-target viruses such as H5N2, H5N3, H7N2, H1N1, and H2N2. This aptasensor provides a foundation for the development of a portable aptasensor instrument.