• Journal of Microbiology
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• 제43권spc1호
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• pp.110-117
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• 2005

Salmonella enterica serovar Typhimurium causes human gastroenteritis and a systemic typhoid-like infection in mice. A critical virulence determinant of Salmonella is the ability to invade mammalian cells. The expression of genes required for invasion is tightly regulated by environmental conditions and a variety of regulatory genes. The hilA regulator encodes an OmpR/ToxR family transcriptional regulator that activates the expression of invasion genes in response to both environmental and genetic regulatory factors. Work from several laboratories has highlighted that regulation of hilA expression is a key point for controlling expression of the invasive phenotype. A number of positive regulators of hilA expression have been identified including csrAB, sirA/barA, pstS, hilC/sirC/sprA, fis, and hilD. HilD, an AraC/XylS type transcriptional regulator, is of particular importance as a mutation in hilD results in a 14-fold decrease in chromosomal hilA::Tn5lacZY-080 expression and a 53-fold decrease in invasion of HEp-2 cells. It is believed that HilD directly regulates hilA expression as it has been shown to bind to hilA promoter sequences. In addition, our research group, and others, have identified genes (hilE, hha, pag, and lon) that negatively affect hilA transcription. HilE appears to be an important Salmonella-specific regulator that plays a critical role in inactivating hilA expression. Recent work in our lab has been directed at understanding how environmental signals that affect hilA expression may be processed through a hilE pathway to modulate expression of hilA and the invasive phenotype. The current understanding of this complex regulatory system is reviewed.

• 한국미생물·생명공학회지
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• 제40권1호
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• pp.70-75
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• 2012

살모넬라(Salmonella)의 염색체에 존재하는 병원성 유전자의 집합체인 Salmonella pathogenicity island(SPI)1 과 2는 살모넬라가 유발하는 다양한 질병에 중요한 역할을 한다. SPI1의 발현을 유도하는 HilD는 Luria-Bertani(LB) 배지 조건에서 SPI2의 발현 활성인자로 작용하는 것으로 알려져 있으나 LB 배지 내에서 hilD 유전자의 발현 양상은 아직까지 연구되지 않았다. 본 연구에서는 LB 배지에 살모넬라를 배양하면서 hilD 유전자의 발현과 단백질 양을 조사하였으며 SPI2 유전자인 sseA의 발현과 비교하였다. hilD의 발현은 대수 증식기 경과 후 정지기(stationary phase)로 전환되는 시기에 비약적으로 증가하였으나 sseA의 발현은 정지기 후반부에 최대로 증가하였다. 즉, 후반 정지기에서 HilD 단백질은 낮은 수준으로 존재함에도 불구하고 SPI2의 발현을 유도한다는 것을 알 수 있었다. SPI1의 다른 발현 조절인자인 hilA와 invF의 변이체에서 sseA의 발현을 살펴본 결과 invF의 변이는 hilD와는 다르게 배지 조건에 상관없이 오히려 sseA의 발현을 증가시켰다. 또한, InvF의 과발현은 sseA 발현을 정상 수준으로 복원시켰지만 추가적인 감소는 일으키지 않는다는 것을 알 수 있었다. SPI1은 HilD를 이용하여 SPI2의 발현을 유도하지만 반대로 InvF를 이용하여 발현을 억제하기도 하는 이중적인 조절 기전을 가지고 있는 것으로 판단된다.

• Food Science and Biotechnology
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• 제16권5호
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• pp.778-782
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• 2007

This study assessed the effects of the antimicrobial substances sulforaphane, grapefruit seed extracts (GSE), and reuterin on the expression of Salmonella HilA and InvF virulence gene using a LacZY assay (${\beta}$-galactosidase assay) with hilA:lacZY and invF:lacZY fusion strains of Salmonella typhimurium SL1344. Salmonella was grown for 8 hr at $37^{\circ}C$ in the presence of diluted antimicrobial substances ($2\;{\mu}g/mL$ sulforaphane, $20\{\mu}g/mL$ GSE, and 0.26 mM reuterin) at concentrations that did not inhibit the cellular growth of Salmonella. Sulforaphane inhibited the expression of HilA and InvF by 50-90 and 20-80%, respectively. GSE also inhibited the expression of both genes, but to a lesser degree. Among the 3 antimicrobial substances, reuterin showed the least inhibition, which was abolished after 3-4 hr. None of the antimicrobial substances inhibited the ${\beta}$-galactosidase enzyme activity of S. typhimurium. The assay used in this study represents a very sensitive method for screening bioactive substances that inhibit the expression of virulence genes in Salmonella.

• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.175-182
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• 2005

Abstract Salmonella pathogenicity island 1 (SPI1) gene expression is regulated by many environmental signals such as oxygen, osmolarity, and pH. Here, we examined changes in the expression level of various regulatory proteins encoded within SPI1 in response to three different concentrations of NaCl, using primer extension analysis. Transcription of all the regulatory genes tested was activated most when Salmonella were grown in Luria Broth (LB) containing 0.17 M NaCl. The expression of hilA, invF, and hilD was decreased in the presence of 0.47 M NaCl or in the absence of NaCl, while hilC expression was almost constant regardless of the NaCl concentration when Salmonella were grown to exponential phase under low-oxygen condition. The reduced expression of hilA, invF, and hilD resulted in lower invasion of hilC mutant to the cultured animal cells when the mutant was grown in the presence of 0.47 M NaCl or in the absence of NaCl prior to infection. Among the proteins secreted via the SPI1-type III secretion system (TTSS), the level of sopE2 expression was not influenced by medium osmolarity. Various effects of osmolarity on virulence gene regulation observed in this study is one example of multiple regulatory pathways used by Salmonella to cause infection.

• Genomics & Informatics
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• 제19권3호
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• pp.30.1-30.8
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• 2021

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1664-1672
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• 2013

In Salmonella enterica serovar Typhimurium, many genes encoded within Salmonella pathogenicity island 1 (SPI1) are required to induce intestinal/diarrheal disease. In this study, we compared the expression of four SPI1 genes (hilA, invF, prgH, and sipC) under shaking and standing culture conditions and found that the expression of these genes was highest during the transition from the exponential to stationary phase under shaking conditions. To identify regulators associated with the stationary phase-dependent activation of SPI1, the effects of selected regulatory genes, including relA/spoT (ppGpp), luxS, ihfB, hfq, and arcA, on the expression of hilA and invF were compared under shaking conditions. Mutations in the hfq and arcA genes caused a reduction in hilA and invF expression (more than 2-fold) in the early stationary phase only, whereas the lack of ppGpp and IHF decreased hilA and invF gene expression during the entire stationary phase. We also found that hfq and arcA mutations caused a reduction of hilD expression upon entry into the stationary phase under shaking culture conditions. Taken together, these results suggest that Hfq and ArcA regulate the hilD promoter, causing an accumulation of HilD, which can trigger a stationary phase-dependent activation of SPI1 genes under shaking culture conditions.

• 한국식품영양과학회지
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• 제26권4호
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• pp.669-674
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• 1997

면역활성, 항균성 등의 기능성을 보여 식품첨가물로 전량 수입에 의존하여 사용되는 human lactoferrin을 진핵세포에서의 생산을 시도하였다. 우선, 항균성을 보이는 lactoferrin에 대하여 생육저해가 없는 host cell에 lactoferrin 유전자를 발현시키고자 lactoferrin에 대한 항균력을 실험한 결과 Pichia pastoris는 생육저해를 일으키지 않아 이를 lactoferrin 생산균주로 선정하였다. Pichia를 숙주로 하는 pHIL-SI expression vector에 lactoferrin 유전자를 삽입 하였을 때 genomic DNA에 유전자가 integration 되었다. 즉, transformant JY-1, JY-2는 PCR(polymerase chain reaction)과 southern blotting에 의하여 2.4Kb의 크기의 HLF(human lactoferrin) 유전자가 삽입되었음을 확인하였다. 유전자 발현을 검토한 결과 transformant JY-1는 immunoblotting에 의하여 lactoferrin 단백질 생산을 확인하였다. 배양시간에 따른 HLF의 생산성을 알아본 결과 48시간 이후에 75KDa의 HLF단백질이 분비됨을 확인하였다

• 미생물학회지
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• 제34권3호
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• pp.108-114
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• 1998

Salmonella typhimurium에서 병독성 발현, 특히 숙주세포내로의 침입에 중요한 역할을 하는 유전자들의 집단(gene cluster)인 Salmonella Pathogenicity Island 2(SPI2)의 발현에 대한 다양한 환경요인들의 영향을 조사하였다. 이를 위해 SPI2의 주요 유전자인 ssaJ와 ssaK의 promoter를 포함하는 regulatory region을 promoterless lac operon과 융합시켜 reporter를 제조하였다. 그리고 산소농도, 삼투압, pH, 탄소원 결핍 및 glycerol 참가 등 여러 환경요인들의 변화가 이 reporter 유전자의 발현에 미치는 효과를 조사한 결과 저산소, 낮은 삼투압, 약 알칼리 등이 ssaJ와 ssaK의 발현을 증가시켰으며 위의 세조건이 함께 존재할 때 그 발현이 가장 크게 증가하는 것으로 나타났다. 그러나 탄소원 결핍이나 glycerol 첨가는 이 두 유전자들의 발현에 영향을 주지 않았다. 또한 이상과 같은 환경인자들의 효과는 S. typhimurium의 세 가지 야생형인 LT2, UK1과 SL1344 모두에서 동일한 양상을 보였다. 다른 한편 SPI1의 transcriptional activator를 암호화하는 조절유전자인 hilA의 돌연변이는 ssaJ와 ssaK의 발현에 영향을 미치지 않음도 밝혀냈다. 따라서 이상의 결과는 SPI1과 SPI2가 서로 별개의 조절계에 의해 그 발현이 조절됨을 보여준다.

• 한국미생물·생명공학회지
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• 제51권4호
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• pp.390-402
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• 2023

Salmonella are Gram-negative pathogenic bacteria commonly found in food animals such as poultry and swine and potentially constitute risks and threats to food safety and public health through transmissible virulence and antimicrobial resistance (AMR) genes. Although there are previous studies in the Philippines regarding genotypic and phenotypic AMR in Salmonella, there are very few on virulence and their associations. Hence, this study collected 700 Salmonella isolates from swine samples in abattoirs and wet markets among four districts in Metro Manila and characterized their genotypic virulence and β-lactam AMR profiles. Gene frequency patterns and statistical associations between virulence and bla genes and comparisons based on location types (abattoirs and wet markets) and districts were also determined. High prevalence (>50%) of virulence genes was detected encompassing Salmonella pathogenicity islands (SPIs) 1-5 suggesting their pathogenic potential, but none possessed plasmid-borne virulence genes spvR and spvC. For bla, bla_TEM was detected with high prevalence (>45%) and revealed significant associations to four SPI genes, namely, avrA, hilA, mgtC, and spi4R, which suggest high resistance potential particularly to β-lactam antibiotics and relationships with pathogenicity that remain mechanistically unestablished until now. Lastly, comparisons of location types and districts showed variations in gene prevalence suggesting effects from environmental factors throughout the swine production chain. This study provides vital data on the genotypic virulence and AMR of Salmonella from swine in abattoirs and wet markets that suggest their pathogenicity and resistance potential for policymakers to implement enforced surveillance and regulations for the improvement of the Philippine swine industry.

• Molecules and Cells
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• 제28권4호
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• pp.389-395
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• 2009

We previously observed that the transcription of some flagellar genes decreased in Salmonella Typhimurium tdcA mutant, which is a gene encoding the transcriptional activator of the tdc operon. Since flagella-mediated bacterial motility accelerates the invasion of Salmonella, we have examined the effect of tdcA mutation on the invasive ability as well as the flagellar biosynthesis in S. Typhimurium. A tdcA mutation caused defects in motility and formation of flagellin protein, FliC in S. Typhimurium. Invasion assays in the presence of a centrifugal force confirmed that the defect of flagellum synthesis decreases the ability of Salmonella to invade into cultured epithelial cells. In addition, we also found that the expression of Salmonella pathogenicity island 1 (SPI1) genes required for Salmonella invasion was down-regulated in the tdcA mutant because of the decreased expression of fliZ, a positive regulator of SPI1 transcriptional activator, hilA. Finally, the virulence of a S. Typhimurium tdcA mutant was attenuated compared to a wild type when administered orally. This study implies the role of tdcA in the invasion process of S. Typhimurium.