TamAB is regulated by PhoPQ and functions in outer membrane homeostasis during Salmonella pathogenesis.

Salmonella survive and replicate in macrophages, which normally kill bacteria by exposing them to a variety of harsh conditions and antimicrobial effectors, many of which target the bacterial cell envelope. The PhoPQ two-component system responds to the phagosome environment and induces factors that protect the outer membrane, allowing adaptation and growth in the macrophage. We show that PhoPQ induces the transcription of the tamAB operon both in vitro and in macrophages. The TamA protein is structurally similar to BamA, an essential protein in the Bam complex that assembles ß-barrel proteins in the outer membrane, while TamB is an AsmA-family protein implicated in lipid transport between the inner and outer membranes. We show that the Bam machinery is stressed in vitro under low Mg2+, low pH conditions that mimic the phagosome. Not surprisingly, mutations affecting Bam function confer significant virulence defects. Although loss of TamAB alone confers no virulence defect, a tamAB deletion confers a synthetic phenotype in bam mutant backgrounds in animals and macrophages, and in vitro upon treatment with vancomycin or sodium dodecyl sulfate. Mutations affecting YhdP, which functions in partial redundancy with TamB, also confer synthetic phenotypes with bam mutations in the animal, but this interaction is not evident in vitro. Thus, in the harsh phagocytic environment of the macrophage, the outer membrane Bam machinery is compromised, and the TamAB system, and perhaps other PhoPQ-regulated factors, is induced to compensate. It is most likely that TamAB and other systems assist the Bam complex indirectly by affecting outer membrane properties. IMPORTANCE The TamAB system has been implicated in both outer membrane protein localization and phospholipid transport between the inner and outer membranes. We show that the ß-barrel protein assembly complex, Bam, is stressed under conditions thought to mimic the macrophage phagosome. TamAB expression is controlled by the PhoPQ two-component system and induced in macrophages. This system somehow compensates for the Bam complex as evidenced by the fact that mutations affecting the two systems confer synthetic phenotypes in animals, macrophages, and in vitro in the presence of vancomycin or SDS. This study has implications concerning the role of TamAB in outer membrane homeostasis. It also contributes to our understanding of the systems necessary for Salmonella to adapt and reproduce within the macrophage phagosome.

Small RNAs Activate Salmonella Pathogenicity Island 1 by Modulating mRNA Stability through the hilD mRNA 3' Untranslated Region.

Salmonella enterica serovar Typhimurium is an enteric pathogen associated with foodborne disease. Salmonella invades the intestinal epithelium using a type three secretion system encoded on Salmonella pathogenicity island 1 (SPI-1). SPI-1 genes are tightly regulated by a complex feed-forward loop to ensure proper spatial and temporal expression. Most regulatory input is integrated at HilD, through control of hilD mRNA translation or HilD protein activity. The hilD mRNA possesses a 310-nucleotide 3' untranslated region (UTR) that influences HilD and SPI-1 expression, and this regulation is dependent on Hfq and RNase E, cofactors known to mediate small RNA (sRNA) activities. Thus, we hypothesized that the hilD mRNA 3' UTR is a target for sRNAs. Here, we show that two sRNAs, SdsR and Spot 42, regulate SPI-1 by targeting different regions of the hilD mRNA 3' UTR. Regulatory activities of these sRNAs depended on Hfq and RNase E, in agreement with previous roles found for both at the hilD 3' UTR. Salmonella mutants lacking SdsR and Spot 42 had decreased virulence in a mouse model of infection. Collectively, this work suggests that these sRNAs targeting the hilD mRNA 3' UTR increase hilD mRNA levels by interfering with RNase E-dependent mRNA degradation and that this regulatory effect is required for Salmonella invasiveness. Our work provides novel insights into mechanisms of sRNA regulation at bacterial mRNA 3' UTRs and adds to our knowledge of post-transcriptional regulation of the SPI-1 complex feed-forward loop. IMPORTANCE Salmonella enterica serovar Typhimurium is a prominent foodborne pathogen, infecting millions of people a year. To express virulence genes at the correct time and place in the host, Salmonella uses a complex regulatory network that senses environmental conditions. Known for their role in allowing quick responses to stress and virulence conditions, we investigated the role of small RNAs in facilitating precise expression of virulence genes. We found that the 3' untranslated region of the hilD mRNA, encoding a key virulence regulator, is a target for small RNAs and RNase E. The small RNAs stabilize hilD mRNA to allow proper expression of Salmonella virulence genes in the host.

Cation Homeostasis: Coordinate Regulation of Polyamine and Magnesium Levels in Salmonella.

Polyamines are organic cations that are important in all domains of life. Here, we show that in Salmonella, polyamine levels and Mg2+ levels are coordinately regulated and that this regulation is critical for viability under both low and high concentrations of polyamines. Upon Mg2+ starvation, polyamine synthesis is induced, as is the production of the high-affinity Mg2+ transporters MgtA and MgtB. Either polyamine synthesis or Mg2+ transport is required to maintain viability. Mutants lacking the polyamine exporter PaeA, the expression of which is induced by PhoPQ in response to low Mg2+, lose viability in the stationary phase. This lethality is suppressed by blocking either polyamine synthesis or Mg2+ transport, suggesting that once Mg2+ levels are reestablished, the excess polyamines must be excreted. Thus, it is the relative levels of both Mg2+ and polyamines that are regulated to maintain viability. Indeed, sensitivity to high concentrations of polyamines is proportional to the Mg2+ levels in the medium. These results are recapitulated during infection. Polyamine synthesis mutants are attenuated in a mouse model of systemic infection, as are strains lacking the MgtB Mg2+ transporter. The loss of MgtB in the synthesis mutant background confers a synthetic phenotype, confirming that Mg2+ and polyamines are required for the same process(es). Mutants lacking PaeA are also attenuated, but deleting paeA has no phenotype in a polyamine synthesis mutant background. These data support the idea that the cell coordinately controls both the polyamine and Mg2+ concentrations to maintain overall cation homeostasis, which is critical for survival in the macrophage phagosome. IMPORTANCE Polyamines are organic cations that are important in all life forms and are essential in plants and animals. However, their physiological functions and regulation remain poorly understood. We show that polyamines are critical for the adaptation of Salmonella to low Mg2+ conditions, including those found in the macrophage phagosome. Polyamines are synthesized upon low Mg2+ stress and partially replace Mg2+ until cytoplasmic Mg2+ levels are restored. Indeed, it is the sum of Mg2+ and polyamines in the cell that is critical for viability. While Mg2+ and polyamines compensate for one another, too little of both or too much of both is lethal. After cytoplasmic Mg2+ levels are reestablished, polyamines must be exported to avoid the toxic effects of excess divalent cations.

The Small RNA MicC Downregulates hilD Translation To Control the Salmonella Pathogenicity Island 1 Type III Secretion System in Salmonella enterica Serovar Typhimurium.

Salmonella enterica serovar Typhimurium invades the intestinal epithelium and induces inflammatory diarrhea using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). Expression of the SPI1 T3SS is controlled by three AraC-like regulators, HilD, HilC, and RtsA, which form a feed-forward regulatory loop that leads to activation of hilA, encoding the main transcriptional regulator of the T3SS structural genes. This complex system is affected by numerous regulatory proteins and environmental signals, many of which act at the level of hilD mRNA translation or HilD protein function. Here, we show that the sRNA MicC blocks translation of the hilD mRNA by base pairing near the ribosome binding site. MicC does not induce degradation of the hilD message. Our data indicate that micC is transcriptionally activated by SlyA, and SlyA feeds into the SPI1 regulatory network solely through MicC. Transcription of micC is negatively regulated by the OmpR/EnvZ two-component system, but this regulation is dependent on SlyA. OmpR/EnvZ control SPI1 expression partially through MicC but also affect expression through other pathways, including an EnvZ-dependent, OmpR-independent mechanism. MicC-mediated regulation plays a role during infection, as evidenced by an SPI1 T3SS-dependent increase in Salmonella fitness in the intestine in the micC deletion mutant. These results further elucidate the complex regulatory network controlling SPI1 expression and add to the list of sRNAs that control this primary virulence factor. IMPORTANCE The Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS) is the primary virulence factor required for causing intestinal disease and initiating systemic infection. The system is regulated in response to a large variety of environmental and physiological factors such that the T3SS is expressed at only the appropriate time and place in the host during infection. Here, we show how the sRNA MicC affects expression of the system. This work adds to our detailed mechanistic studies aimed at a complete understanding of the regulatory circuit.

PaeA (YtfL) protects from cadaverine and putrescine stress in Salmonella Typhimurium and E. coli.

Salmonella and E. coli synthesize, import, and export cadaverine, putrescine, and spermidine to maintain physiological levels and provide pH homeostasis. Both low and high intracellular levels of polyamines confer pleiotropic phenotypes or lethality. Here, we demonstrate that the previously uncharacterized inner membrane protein PaeA (YtfL) is required for reducing cytoplasmic cadaverine and putrescine concentrations. We identified paeA as a gene involved in stationary phase survival when cells were initially grown in acidic medium, in which they produce cadaverine. The paeA mutant is also sensitive to putrescine, but not to spermidine or spermine. Sensitivity to external cadaverine in stationary phase is only observed at pH > 8, suggesting that the polyamines need to be deprotonated to passively diffuse into the cell cytoplasm. In the absence of PaeA, intracellular polyamine levels increase and the cells lose viability. Degradation or modification of the polyamines is not relevant. Ectopic expression of the known cadaverine exporter, CadB, in stationary phase partially suppresses the paeA phenotype, and overexpression of PaeA in exponential phase partially complements a cadB mutant grown in acidic medium. These data support the hypothesis that PaeA is a cadaverine/putrescine exporter, reducing potentially toxic levels under certain stress conditions.

Oxygen-dependent regulation of SPI1 type three secretion system by small RNAs in Salmonella enterica serovar Typhimurium.

Salmonella Typhimurium induces inflammatory diarrhea and uptake into intestinal epithelial cells using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). Three AraC-like regulators, HilD, HilC and RtsA, form a feed-forward regulatory loop that activates transcription of hilA, encoding the activator of the T3SS structural genes. Many environmental signals and regulatory systems are integrated into this circuit to precisely regulate SPI1 expression. A subset of these regulatory factors affects translation of hilD, but the mechanisms are poorly understood. Here, we identified two sRNAs, FnrS and ArcZ, which repress hilD translation, leading to decreased production of HilA. FnrS and ArcZ are oppositely regulated in response to oxygen, one of the key environmental signals affecting expression of SPI1. Mutational analysis demonstrates that FnrS and ArcZ bind to the hilD mRNA 5' UTR, resulting in translational repression. Deletion of fnrS led to increased HilD production under low-aeration conditions, whereas deletion of arcZ abolished the regulatory effect on hilD translation aerobically. The fnrS arcZ double mutant has phenotypes in a mouse oral infection model consistent with increased expression of SPI1. Together, these results suggest that coordinated regulation by these two sRNAs maximizes HilD production at an intermediate level of oxygen.

Intestinal Long-Chain Fatty Acids Act as a Direct Signal To Modulate Expression of the Salmonella Pathogenicity Island 1 Type III Secretion System.

UNLABELLED: Salmonella enterica serovar Typhimurium uses the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS) to induce inflammatory diarrhea and bacterial uptake into intestinal epithelial cells. The expression of hilA, encoding the transcriptional activator of the T3SS structural genes, is directly controlled by three AraC-like regulators, HilD, HilC, and RtsA, each of which can activate hilD, hilC, rtsA, and hilA genes, forming a complex feed-forward regulatory loop. Expression of the SPI1 genes is tightly controlled by numerous regulatory inputs to ensure proper timing in production of the T3SS apparatus. Loss of FadD, an acyl coenzyme A (acyl-CoA) synthetase required for degradation of long-chain fatty acids (LCFAs), was known to decrease hilA expression. We show that free external LCFAs repress expression of hilA independently of FadD and the LCFA degradation pathway. Genetic and biochemical evidence suggests that LCFAs act directly to block primarily HilD activity. Further analyses show that in the absence of FadD, hilA expression is downregulated due to endogenous production of free LCFAs, which are excreted into the culture medium via TolC and then transported back into the bacterial cell via FadL. A fadL mutant is more virulent than the wild-type strain in mouse oral competition assays independently of LCFA degradation, showing that, in the host, dietary LCFAs serve as a signal for proper regulation of SPI1 expression, rather than an energy source. IMPORTANCE: To cause disease, Salmonella must respond to diverse environmental cues to express its invasion machinery at the appropriate location in the host intestine. We show that host intestinal free long-chain fatty acids (LCFAs) affect Salmonella invasion by reducing expression of the SPI1 type III secretion system, acting primarily via the AraC-like activator HilD. Degradation of LCFAs is not required for this regulation, showing that free LCFAs serve as a cue to proper intestinal localization to invade host epithelial cells and not as a nutrient source.

Twin-arginine translocation system (tat) mutants of Salmonella are attenuated due to envelope defects, not respiratory defects.

The twin-arginine translocation system (Tat) transports folded proteins across the cytoplasmic membrane and is critical to virulence in Salmonella and other pathogens. Experimental and bioinformatic data indicate that 30 proteins are exported via Tat in Salmonella Typhimurium. However, there are no data linking specific Tat substrates with virulence. We inactivated every Tat-exported protein and determined the virulence phenotype of mutant strains. Although a tat mutant is highly attenuated, no single Tat-exported substrate accounts for this virulence phenotype. Rather, the attenuation is due primarily to envelope defects caused by failure to translocate three Tat substrates, the N-acetylmuramoyl-l-alanine amidases, AmiA and AmiC, and the cell division protein, SufI. Strikingly, neither the amiA amiC nor the sufI mutations alone conferred any virulence defect. Although AmiC and SufI have previously been localized to the divisome, the synthetic phenotypes observed are the first to suggest functional overlap. Many Tat substrates are involved in anaerobic respiration, but we show that a mutant completely deficient in anaerobic respiration retains full virulence in both the oral and systemic phases of infection. Similarly, an obligately aerobic mutant is fully virulent. These results suggest that in the classic mouse model of infection, S. Typhimurium is replicating only in aerobic environments.

Integrating global regulatory input into the Salmonella pathogenicity island 1 type III secretion system.

Salmonella enterica serovar Typhimurium uses the Salmonella pathogenicity island 1 (SPI1) type III secretion system to induce inflammatory diarrhea and bacterial uptake into intestinal epithelial cells. The expression of hilA, encoding the transcriptional activator of the SPI1 structural genes, is directly controlled by three AraC-like regulators, HilD, HilC, and RtsA, each of which can activate the hilD, hilC, rtsA, and hilA genes, forming a complex feed-forward regulatory loop. A large number of factors and environmental signals have been implicated in SPI1 regulation. We have developed a series of genetic tests that allows us to determine where these factors feed into the SPI1 regulatory circuit. Using this approach, we have grouped 21 of the known SPI1 regulators and environmental signals into distinct classes on the basis of observed regulatory patterns, anchored by those few systems where the mechanism of regulation is best understood. Many of these factors are shown to work post-transcriptionally at the level of HilD, while others act at the hilA promoter or affect all SPI1 promoters. Analysis of the published transcriptomic data reveals apparent coregulation of the SPI1 and flagellar genes in various conditions. However, we show that in most cases, the factors that affect both systems control SPI1 independently of the flagellar protein FliZ, despite its role as an important SPI1 regulator and coordinator of the two systems. These results provide a comprehensive model for SPI1 regulation that serves as a framework for future molecular analyses of this complex regulatory network.

FliZ regulates expression of the Salmonella pathogenicity island 1 invasion locus by controlling HilD protein activity in Salmonella enterica serovar typhimurium.

A prerequisite for Salmonella enterica to cause both intestinal and systemic disease is the direct injection of effector proteins into host intestinal epithelial cells via a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1). These effector proteins induce inflammatory diarrhea and bacterial invasion. Expression of the SPI1 T3SS is tightly regulated in response to environmental signals through a variety of global regulatory systems. We have previously shown that three AraC-like regulators, HilD, HilC, and RtsA, act in a complex feed-forward regulatory loop to control the expression of the hilA gene, which encodes the direct regulator of the SPI1 structural genes. In this work, we characterize a major positive regulator of this system, the flagellar protein FliZ. Through genetic and biochemical analyses, we show that FliZ posttranslationally controls HilD to positively regulate hilA expression. This mechanism is independent of other flagellar components and is not mediated through the negative regulator HilE or through FliZ-mediated RpoS regulation. We demonstrate that FliZ controls HilD protein activity and not stability. FliZ regulates HilD in the absence of Lon protease, previously shown to degrade HilD. Indeed, it appears that FliZ, rather than HilD, is the most relevant target of Lon as it relates to SPI1 expression. Mutants lacking FliZ are significantly attenuated in their ability to colonize the intestine but are unaffected during systemic infection. The intestinal attenuation is partially dependent on SPI1, but FliZ has additional pleiotropic effects.

Salmonella enterica serovar Typhimurium periplasmic superoxide dismutase SodCI is a member of the PhoPQ regulon and is induced in macrophages.

Salmonella enterica serovar Typhimurium replicates within host macrophages during the systemic stage of infection. In the macrophage, the bacteria must survive the respiratory burst that produces superoxide. Serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases, SodCI and SodCII, but only SodCI contributes to virulence. Although we have shown that this is primarily due to differences in the two proteins, evidence suggests differential regulation of the two genes. Using transcriptional sodCI- and sodCII-lac fusions, we show that sodCII is under the control of the RpoS sigma factor, as was known for the Escherichia coli ortholog, sodC. In contrast, we show that sodCI is transcriptionally controlled by the PhoPQ two-component regulatory system, which regulates an array of virulence genes required for macrophage survival. Introduction of a phoP-null mutation into the sodCI fusion strain resulted in a decrease in transcription and loss of regulation. The sodCI-lac fusion showed high-level expression in a background containing a phoQ constitutive allele. The sodCI gene is induced 15-fold in bacteria recovered from either the tissue culture macrophages or the spleens of infected mice. Induction in macrophages is dependent on PhoP. The sodCII fusion was induced three- to fourfold in macrophages and animals; this induction was unaffected by loss of PhoP. Thus, sodCI, which is horizontally transferred by the Gifsy-2 phage, is regulated by PhoPQ such that it is induced at the appropriate time and place to combat phagocytic superoxide.