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 FliZmediated 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.During the course of infection, Salmonella enterica serovar Typhimurium induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1) (20,51,53,55). The SPI1 locus is a 40-kb island and carries all of the genes for a functional T3SS apparatus, a number of secreted effectors, and the regulatory proteins HilA, HilC, and HilD (34). RtsA, homologous to HilD and HilC, is encoded on a 15-kb island inserted in the tRNA PheU gene (15). HilA is the master SPI1 regulator and directly binds to the promoters and activates expression of the prg-org and inv-spa operons, encoding the components of the apparatus (2,8,10,11,32). The expression of hilA is controlled by a complex feed-forward regulatory loop consisting of HilD, HilC, and RtsA, each of which can independently activate hilA expression (13) (Fig. 1). Of these three, HilD has the predominant role, but apparently, it alone cannot activate SPI1 sufficiently in vivo (13). HilC and RtsA act as amplifiers of the inducing signals. A number of additional regulatory systems also affect SPI1 regulation (1,17). Most of these systems seem to act through HilD (18), suggesting that HilD is the integration point for signal transduction into the SPI1 system. RtsA, HilD, and HilC directly activate dsbA, which encodes a periplasmic disulfide bond oxidoredu...

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.

Background Current clinical guidelines recommend earlier, more intensive breast cancer screening with both MRI and mammography for women with BRCA mutations. Unspecified details of screening schedules are a challenge for implementing guidelines. Methods A Markov Monte Carlo computer model simulated screening in asymptomatic female BRCA1 and BRCA2 mutation carriers. Three dual-modality strategies were compared with digital mammography (DM) alone: 1) DM and MRI alternating at 6-month intervals beginning at age 25 [Alt25], 2) annual MRI beginning at age 25 with alternating DM added at age 30 [MRI25/Alt30], and 3) DM and MRI alternating at 6-month intervals beginning at age 30 [Alt30]. Primary outcomes were quality-adjusted life years (QALYs), lifetime costs (in 2010 USD), and incremental cost-effectiveness ($/QALY gained). Additional outcomes included potential harms of screening, and lifetime costs stratified into component categories (screening and diagnosis, treatment, mortality, and patient time costs). Results All three dual-modality screening strategies increased QALYs and costs. Alt30 screening had the lowest incremental costs per additional QALY gained: (BRCA1: $74,200/QALY; BRCA2: $215,700/QALY). False-positive test results increased substantially with dual-modality screening, occurring more frequently in BRCA2 carriers. Downstream savings in both breast cancer treatment and mortality costs were outweighed by increases in up-front screening and diagnosis costs. Results were most influenced by estimates of breast cancer risk and MRI cost. Conclusions Alternating MRI and DM screening at 6-month intervals beginning at age 30 is a clinically effective approach to applying current guidelines, and is more cost-effective in BRCA1 compared with BRCA2 gene mutation carriers.

Background While breast cancer screening with mammography and MRI is recommended for BRCA mutation carriers, there is no current consensus on the optimal screening regimen. Methods We used a computer simulation model to compare six annual screening strategies [film mammography (FM), digital mammography (DM), FM and magnetic resonance imaging (MRI) or DM and MRI contemporaneously, and alternating FM/MRI or DM/MRI at six-month intervals] beginning at ages 25, 30, 35, and 40, and two strategies of annual MRI with delayed alternating DM/FM to clinical surveillance alone. Strategies were evaluated without and with mammography-induced breast cancer risk, using two models of excess relative risk. Input parameters were obtained from the medical literature, publicly available databases, and calibration. Results Without radiation risk effects, alternating DM/MRI starting at age 25 provided the highest life expectancy (BRCA1: 72.52 years, BRCA2: 77.63 years). When radiation risk was included, a small proportion of diagnosed cancers were attributable to radiation exposure (BRCA1: <2%, BRCA2: <4%). With radiation risk, alternating DM/MRI at age 25 or annual MRI at age 25/delayed alternating DM at age 30 were most effective, depending on the radiation risk model used. Alternating DM/MRI starting at age 25 also had the highest number of false-positive screens/person (BRCA1: 4.5, BRCA2: 8.1). Conclusions Annual MRI at 25/delayed alternating DM at age 30 is likely the most effective screening strategy in BRCA mutation carriers. Screening benefits, associated risks and personal acceptance of false-positive results, should be considered in choosing the optimal screening strategy for individual women.

The type three secretion system (T3SS), encoded in the pathogenicity island 1 (SPI1) locus, mediates the invasion of the host intestinal epithelium. SPI1 expression is dependent upon three AraC-like regulators: HilD, HilC, and RtsA. These regulators act in a complex feed-forward loop to activate each other and , which encodes the activator of the T3SS structural genes. HilD has been shown to be the major integration point of most signals known to activate the expression of the SPI1 T3SS, acting as a switch to control induction of the system. HilE is a negative regulator that acts upon HilD. Here we provide genetic and biochemical data showing that HilE specifically binds to HilD but not to HilC or RtsA. This protein-protein interaction blocks the ability of HilD to bind DNA as shown by both an reporter system and an gel shift assay. HilE does not affect HilD dimerization, nor does it control the stability of the HilD protein. We also investigated the role of HilE during the infection of mice using competition assays. Although deletion of does not confer a phenotype, the mutation does suppress the invasion defect conferred by loss of FliZ, which acts as a positive signal controlling HilD protein activity. Together, these data suggest that HilE functions to restrict low-level HilD activity, preventing premature activation of SPI1 until positive inputs reach a threshold required to fully induce the system. is a leading cause of gastrointestinal and systemic disease throughout the world. The SPI1 T3SS is required for to induce inflammatory diarrhea and to gain access to underlying tissue. A complex regulatory network controls expression of SPI1 in response to numerous physiological inputs. Most of these signals impinge primarily on HilD translation or activity. The system is triggered when HilD activity crosses a threshold that allows efficient activation of its own promoter. This threshold is set by HilE, which binds to HilD to prevent the inevitable minor fluctuations in HilD activity from inappropriately activating the system. The circuit also serves as a paradigm for systems that must integrate numerous environmental parameters to control regulatory output.