Global transcriptional response of Salmonella enterica serovar Typhi to anti-z66 antiserum


  • Editor: Reggie Lo

Correspondence: Xinxiang Huang, Department of Biochemistry and Molecular Biology, School of Medical Technology, Jiangsu University, Zhenjiang, Jiangsu, 212013, China. Tel.: +86 511 85 03 84 01; fax: +86 511 85 01 25 44; e-mail:


Recent studies have shown that flagella may modulate physiological processes by sensing environmental changes in temperature and moisture. When the z66+ strain of Salmonella enterica serovar Typhi (S. Typhi) was exposed to an antiserum against the z66 flagellar antigen, the fljBA operon was deleted from a linear plasmid, leading to the unidirectional flagellar phase variation from FljB to FliC. We hypothesized that flagella may serve as a sensor that responded to the antiserum by altering gene expression and triggering the unidirectional flagellar phase variation. To test this hypothesis, Salmonella genomic DNA microarrays were used to determine the gene expression profile of the z66+ wild-type strain of S. Typhi treated with the anti-z66 antiserum for 30 min. The results showed that expression levels of 187 genes were altered by more than threefold compared with the same strain treated with control serum. The microarray expression patterns of representative genes were validated by reverse transcriptase-PCR. Importantly, no significant changes in gene expression were observed in the fljB:z66 deletion mutant that was similarly treated with the anti-z66 antiserum. To the best of our knowledge, this is the first study to show the global transcriptional response of Salmonella to antiflagellin antiserum.


Flagella play an important role in Salmonella motility. More than 2300 serovars of Salmonella enterica have been identified on the basis of their flagella (H antigen) and lipopolysaccharide (O antigen) (Ewing, 1986). Most S. enterica serovars are biphasic because they possess two flagellin genes, fliC and fljB, located at different chromosomal loci. They alternately express these two genes through a process called ‘phase variation,’ which is mediated by the recombinase Hin, whose gene is located upstream of the fljBA operon. The fljA gene is cotranscribed with fljB and encodes an inhibitor of fliC expression (Yamamoto & Kutsukake, 2006). Therefore, only one of the two flagellin genes is expressed at any given time.

Salmonella enterica serovar Typhi (S. Typhi) is the pathogen responsible for typhoid fever in humans. It was once considered a monophasic strain because it carries only the fliC gene encoding either the full-length H:d antigen or the truncated H:j antigen (Frankel et al., 1989). However, Guinee et al. (1981) discovered a new flagellar antigen in S. Typhi strains collected from Indonesia and named it z66. Subsequently, the z66+ strain was shown to undergo flagellar phase variation from z66 to d/j antigen after induction by an anti-z66 antiserum, while the d/j-positive strains did not revert to z66 upon treatment with an anti-d/j antiserum (Tamura et al., 1988). We previously cloned the gene encoding H:z66 from z66-positive strains and found a gene cluster similar to the fljBA operon in other biphasic S. enterica serovars, but divergent in the upstream region from the hin locus in a typical fljBA operon (Huang et al., 2004). Recently, Baker et al. (2007a, b) demonstrated that the fljBA gene cluster is located in a novel 27-kb linear plasmid named pBSSB1, and the unidirectional flagellar phase variation is caused by the deletion of a 2.7-kb terminal region containing the majority of the fljBA gene cluster. However, the mechanism underlying the truncation is currently unknown.

Recent experiments demonstrated that the flagellum itself could be a sensor. For example, suboptimal external surface hydration was shown to interfere with secretion of flagellin subunits and filament growth through flagellar sensing of changes in environmental moisture (Wang et al., 2005). Additionally, the flagella of S. Typhi can sense shifts in environmental temperature, and modulate flagella motility through conformational changes of the flagellar switch component FliG (Mashimo et al., 2007). In light of these flagellar sensory functions, we hypothesized that the flagella of S. Typhi may also serve as a sensor in response to the anti-z66 antibody to mediate the unidirectional flagellar phase variation through a series of gene regulation events.

Recently, we developed a Salmonella genomic microarray system based on the genomic information of the S. Typhi Ty2 strain (Sheng et al., 2009). Taking advantage of this microarray tool, we analyzed gene expression profiles of the z66+S. Typhi treated with anti-z66 antiserum for 30 min. This study first revealed the global transcriptional response of Salmonella to antiflagellin antiserum.

Materials and methods

Bacterial cultures

The bacterial strain used in this study was S. Typhi GIFU10007, a z66 antigen-positive wild-type strain (Huang et al., 2004). The rabbit anti-z66 antiserum (National Institute of Infectious Disease, Japan) and nonimmune rabbit serum were incubated in water at 56 °C for 30 min to inactivate the complement of these sera, and then purified using the method of ammonium sulfate salting-out. The titer of the purified anti-z66 antiserum is 1 : 40 000. Salmonella enterica serovar Typhi GIFU10007 were incubated in 1 mL Luria–Bertani (LB) broth at 37 °C overnight, and 100 μL was inoculated into 60 mL LB and incubated at 37 °C with shaking for 4 h. The culture was divided into two, with one culture receiving the rabbit anti-z66 antiserum at a 1 : 300 dilution and another receiving normal rabbit serum at the same dilution as the control. Incubation was continued for 30 min after addition of the anti-z66 antiserum. AN fljB:z66 deletion mutant (Huang et al., 2004), which was a nonpolar mutation identified by relative DNA sequencing and was treated with the same anti-z66 antiserum as the control experiment.

RNA extraction and cDNA probe preparation

Total RNAs were extracted using a Qiagen RNaeasy mini kit according to the manufacturer's protocol and treated with RNAse-free DNAse I (Takara) to diminish the trace mixed DNA as described previously (Xu et al., 2008). cDNAs were reverse transcribed from 20 μg of total RNAs using a SuperScript RT III (Invitrogen) kit and used for microarray probe preparation. Each cDNA sample was individually labeled with cy3- and cy5-conjugated dCTP as described previously (Huang et al., 2007; Sheng et al., 2009), in two separate reactions, with dye swapping. Labeled cDNAs were purified using a QIAquick purification kit (Qiagen) according to the manufacturer's instructions.

Hybridization and scanning

The purified cDNA probes from two different treatments with different fluorescent dye labels were paired and combined, and hybridization was performed as described previously (Sheng et al., 2009). The slides were scanned with a GenePix Personal 4100A scanner (Molecular Devices) under two channels of the appropriate lasers. Each experiment was repeated independently three times, and hence three different pairs of total RNA samples were obtained, and six labeled cDNA probe pairs were generated by dye swapping. Because each gene was also duplicated on each microarray slide, all genes were analyzed as 12 replicates.

Data analysis

genepix pro6.0 (Molecular Devices) was used to quantify the spot intensity. The densitometric values of the spots with DNA sequences representing ORFs were normalized with the average overall intensity of the slide by the global normalization mode. Data were exported into microsoft excel for subsequent analysis as described previously (Sheng et al., 2009), with minor modifications. In brief, the ratio of fluorescent intensities from two channels was first calculated for each individual spot on each slide, and then the average intensity ratio of the same gene from different slides was subsequently derived to represent a mean value of difference in gene expression. This was expressed as log2 (ratio) and entered as one spot in the gene expression profile plot view. Only genes that displayed at least eight valid values in the 12 replicate analyses were subjected to further analysis. The threshold log ratio for differential expression was set at 1.58 to represent greater than threefold changes in the expression levels.

Reverse transcriptase (RT)-PCR validation

RNA purification and reverse transcription were performed as above. Gene-specific primers used for PCR are listed in Table 1. For the reverse transcription, each 20-μL reaction contained 2 μg of total RNA and 10 nmol of specific reverse primers. One microliter of reverse transcription product was subjected to a quantitative PCR assay as described previously (Xu et al., 2008). Each experiment was performed with four RNA samples obtained from four independent experiments.

Table 1.   RT-PCR primers
GenesForward primersReverse primers


Microarrays containing 4157 S. Typhi genes were subjected to genomic transcriptional profiling. Five hundred and four genes with a fluorescent intensity below the 300 cut-off value on most of the slides were filtered out and excluded from analysis. The overall expression profile of the remaining 3653 genes in S. Typhi induced by the anti-z66 antiserum for 30 min is shown in Fig. 1. Analyses of triplicate experiments showed that 187 genes displayed significant differential expression (threefold or higher) between the antiserum-induced and the noninduced (normal rabbit serum) samples. One hundred and twenty-two and 65 genes were up- and downregulated, respectively (Supporting Information, Table S1). While many have unknown functions, the remaining genes were categorized based on their known or putative functions. Many upregulated genes are involved in protein translation, and nucleic acid and ribosomal protein biosynthesis. In contrast to the largely anabolic functions of the upregulated genes, many downregulated genes are associated with catabolism of carbohydrates, amino acids and fatty acids.

Figure 1.

 DNA microarray analysis of anti-z66 antiserum-induced gene expression regulation. Red circles correspond to genes whose expression was upregulated by more than threefold. Blue circles correspond to genes whose expression was not significantly altered. Yellow circles correspond to genes whose expression was downregulated by more than threefold.

To verify the microarray results, expression levels of several representative genes including fis, fliC, rplB, purB, acs and poxB were also investigated by RT-PCR. As shown in Fig. 2, there was significant concordance between the RT-PCR results and the microarray analysis. Taken together, these results indicate that upon induction with the anti-z66 antiserum, cells undergo broad regulation of gene expression to promote synthesis of nucleic acids and proteins and concurrently suppress the breakdown of the building blocks.

Figure 2.

 RT-PCR validation of the microarray results. Error bars represent SD from the mean values of replicated data. There was a significant concordance between the RT-PCR results and the microarray analysis. Ab, antibody; WT, wild type.

To investigate whether such broad regulation requires flagella, we next analyzed gene expression patterns of a fljB:z66 deletion mutant treated in the same manner as the wild-type strain. Remarkably, we did not observe similar gene expression changes in this flagella-free strain (Fig. 3), strongly suggesting that the observed gene expression regulation is flagella dependent.

Figure 3.

 Comparison of global transcriptional responses of z66+ wild-type strain and fljB:z66-deleted mutant to anti-z66 antiserum. Blue circles and pink circles represent gene expressional regulations in the fljB:z66-deleted mutant and z66-positive wild-type strain, respectively. None of the significant gene expression changes (over threefold) in wild-type strain was found in the fljB:z66-deleted mutant. Ab, antibody; WT, wild type.


The flagella of bacteria were traditionally considered only as the motility apparatus. In particular, the flagella of Salmonella have been implicated previously in the invasion of host cells (Jones et al., 1992). Conversely, the flagellin subunit is an important target for pathogen recognition by the mammalian innate immune system through Toll-like receptor -5 (Hayashi et al., 2001). Therefore, antibodies against flagellin constitute an adverse environmental factor for bacteria. Given the sensory function of flagella of Salmonella in response to temperature and moisture, we hypothesized that upon challenge by the antibody, the flagellum of S. Typhi might have a sensory function in response to the antiflagellin antibody. In the current study, genomic transcriptional analysis showed that S. Typhi has a wide array of responses to the antiflagellin antibody stress at the gene transcription level. The results of selected quantitative RT-PCR and the fact that many of the 187 genes with significantly altered expression are units of operons and genes of the same operon tend to be coordinately regulated also validate our microarray data. Meanwhile, none of the similar significant gene expression changes were found in the fljB:z66 mutant treated with the antiflagellin antibody in the same manner as the wild-type strain. We also performed the similar experiments with another kind of anti-z66 antibody prepared by immunizing rabbits with a z66-specific flagellin that was expressed in Escherichia coli JM109 with an fljB:z66 recombinant plasmid (pET28a-fljB) and purified with a Ni-TED packed column, and obtained very similar results (data not shown). These results suggest that the transcriptional responses of S. Typhi are H:z66 flagellum dependent.

When S. Typhi was treated with anti-z66 antiserum for 30 min, significant induction was observed for many genes involved in DNA replication, transcription and translation. These genes include recO, priB, nusA, pheS, pheT, infA, prfA, prfC, fusA, trmD, yfjA, rnpA, rph and rhlE. Their expression was increased by more than threefold. Most remarkably, 44 genes encoding ribosomal proteins were coordinately upregulated. Moreover, expression of several genes (carAB, guaBA, purM, prsA and purB) involved in nucleotide biosynthesis pathways increased significantly in our experiments. In addition, genes (speD and potABC) involved in the biosynthesis and transport of spermidine, a kind of polyamine known to be necessary for cell growth, were also upregulated in the responses. Polyamines are reported to play an important role in the bacterial responses to temperature shock, oxidative stress, choline limitation and in vivo growth in Streptococcus pneumoniae pathogenesis (Shah et al., 2008). Taken together, this suggests that upon treatment with the anti-z66 antibody, S. Typhi become actively involved in robust DNA, RNA and protein biosynthesis.

Transcriptional expression changes of genes associated with the metabolism of substrates often emerge in bacterial responses to environmental stresses (Dressaire et al., 2008; Hüfner et al., 2008). In this study, we also observed different responses in genes involved in energy metabolism. On the one hand, genes involved in transport and metabolism of carbohydrates, amino acids and fatty acids (pfkB, poxB, lldP, aldB, acs, ugpBA, fadBA, dadA, argDT, astD, gltJKL and gabDT) were significantly downregulated. On the other, several genes involved in oxidative phosphorylation and energy production (ydgQ, rnfD, cydBA and atpACDG) were upregulated. Of course, we are also interested in the expression of flagellar genes in this response. Surprisingly, genes involved in flagellar biosynthesis and function exhibited no significant differential expression, except for the flagellin gene fliC (reduced by more than threefold). An fliC expression repress gene, fljA, is located downstream of fljB:z66 on the linear plasmid (Baker et al., 2007a, b; Zou et al., 2009). We did not find the truncation of the linear plasmid in the initial stage of the antiflagellum antibody stress. The essential relationship between this early transcriptional responses and the later phase variation of S. Typhi by truncating the linear plasmid requires more experiments for clarification.

In summary, this study shows that the global transcriptional response of Salmonella to antiflagellin antiserum are flagellum dependent. Without a control experiment, using specific antinonflagellin antibodies at this time, we cannot completely exclude the possibility that some gene expression regulation of the response may appear in responses to attacks by other kinds of antibodies.


We thank Professor T. Ezaki (Gifu University) for the bacterial strain, anti-z66 antiserum and continuous support. This work was supported by the National Natural Science Foundation of China (30870095), the National Special Scientific Program (2008ZX10004-009) and the National Key Technology R&D Program (2006BAK02A15).