response
Induction of oxidative stress resulted in the lowest number of differentially expressed genes compared to other RNA-‐Seq experiments. This is likely the result of a targeted bacterial response to this environmental stress. Catalase, which is a member of the OxyR regulon in the 86-‐028NP strain, was previously
shown to be responsible for scavenging high concentrations of hydrogen peroxide in H. influenzae (Pauwels et al., 2004, Harrison et al., 2007). A very high up-‐regulation of the catalase gene, hktE, in this study implies that the majority of hydrogen peroxide was scavenged by catalase in both Rd and R2866. This supports the idea that oxidative stress led to a more targeted and straightforward response than other tested infection-‐relevant conditions in this study. As the oxyR gene is an important mediator of oxidative stress defence in H. influenzae, it was surprising that there was no change in oxyR expression in this study. It is possible that the changes of the oxyR mRNA transcript levels were rapid and transient. As observed in a study by Whitby et al., oxyR transcript levels returned to normal levels within 10 minutes after treatment with hydrogen peroxide (Whitby et al., 2012).
A total of 11 members of the oxyR regulon were previously identified in the 86-‐ 028NP strain of H. influenzae, all of which were up-‐regulated during oxidative stress in a microarray study (Harrison et al., 2007). Of these, hktE, gnd, pgdX and dps were up-‐regulated in both Rd and R2866 during oxidative stress in this study. An OxyR-‐regulated gene, NTHI0684, in the 86-‐028NP strain, encoding a hypothetical protein, was also up-‐regulated in both strains in this study. Interestingly, it coded for a putative membrane protein in Rd and a putative CRISPR-‐associated protein, Cas2, in R2866. Sequence search on the InterPro database revealed no homology to known protein families or domains. Remaining members of the OxyR regulon were genes pntA and pntB, up-‐ regulated in Rd only, as well as the yfeABCD locus, the expression of which was not induced in either of the strains in this study. This is different to a microarray study by Whitby et al., where the yfeB gene was up-‐regulated in Rd in response to hydrogen peroxide, while gnd was not (Whitby et al., 2012). The yfeABCD locus was also highly up-‐regulated in response to oxidative stress in another microarray study by Harrison et al. (Harrison et al., 2007). While there are clear inter-‐strain variations as well as differences between study designs, the overall OxyR-‐dependent response to oxidative stress seems to be similar among H. influenzae strains.
Several iron acquisition and ferritin-‐like protein genes were up-‐regulated during oxidative stress as well. Ferritin-‐like proteins sequester ferrous iron, thus preventing further oxidative damage through Fenton reaction. The up-‐ regulation of iron uptake could be utilised for the repair of damaged iron-‐ sulphur cluster proteins. Iron-‐sulphur clusters are present as cofactors in a large number of enzymes, mediating a variety of different roles in bacteria (Yoch and Carithers, 1979). There was up-‐regulation of genes associated with iron-‐sulphur cluster formation in both strains as well. The iron acquisition gene locus, hxuABC, as well as genes tbp1, tbp2, hitA and hemR, up-‐regulated in R2866 only, were shown to be part of the Fur regulon in the 86-‐028NP strain, suggesting that this transcriptional regulator plays a role in oxidative stress defence in R2866 as well. Iron homeostasis, including transcriptional regulation by Fur, plays an important role during oxidative stress in H. influenzae as shown in this and previous studies (Harrison et al., 2015).
Genes recA, lexA, recX and ruvA, up-‐regulated in both strains during oxidative stress, were previously shown to be involved in the SOS response in H. influenzae as part of the LexA regulon (Sweetman et al., 2005). The expression of genes recA and recN was also shown to be induced in the 86-‐028NP strain in response to hydrogen peroxide (Harrison et al., 2007). Up-‐regulation of these genes, along with multiple other genes with predicted roles in SOS response and protection against DNA damage, highlights the drastic damaging effect that hydrogen peroxide has on bacterial DNA (Rohwer and Azam, 2000).
Up-‐regulation of the acetolactate synthase gene locus, ilvHI, in Rd during oxidative stress was curious. This enzyme, along with an up-‐regulated ketol-‐ acid reductoisomerase gene, ilvC, is involved in the biosynthesis of branched-‐ chain amino acids leucine, isoleucine and valine (Ricca et al., 1988). Branched-‐ chain amino acid supplementation has been shown to decrease oxidative stress levels in eukaryotic cells, though it is not clear whether this translates to prokaryotes as well (Iwasa et al., 2013). In addition, the lack of branched-‐chain amino acids in the host cells induced expression of virulence genes in Listeria monocytogenes (Lobel et al., 2012). Therefore, the concurrent up-‐regulation of
branched-‐chain amino acid biosynthesis during oxidative stress could represent H. influenzae responding to host-‐like conditions.
Although the induced expression of genes during oxidative stress in Rd and R2866 was largely similar, there were a lot more differences in down-‐regulated genes. For instance, R2866 only contained 27 down-‐regulated genes, whereas Rd had 97. In addition, there was no overlap in the ten most highly down-‐ regulated genes between the two strains. It is not clear why the response was this different, but a partial explanation could be that the gene expression of one of the R2866 replicates from the mid-‐exponential group noticeably differed from the other two, as inferred with hierarchical clustering. Therefore, it could result in the underestimation of some differentially expressed genes. However, since the up-‐regulated genes in R2866 had a much greater similarity to Rd, this cannot plausibly be the whole reason. The difference in down-‐regulation could be simply explained by different strategies that R2866 employs during oxidative stress.
As there were a large number of up-‐regulated iron-‐related genes during oxidative stress, down-‐regulation of several other iron-‐associated genes during the same condition highlights the complex dynamic of iron homeostasis and oxidative stress in H. influenzae. Down-‐regulation of several ribosomal protein genes was most likely related to a reduction in protein synthesis resulting from a general response to a stress condition.
The arginine uptake locus, which is part of the Fur regulon in the 86-‐028NP strain, was down-‐regulated during oxidative stress in the Rd strain. In agreement with that, there was also down-‐regulation of other gene clusters that were Fur-‐regulated in the 86-‐028NP strain. This included DMSO and nitrite reductase loci as well as the hbpA gene (Harrison et al., 2013). Genes ftnA1 and ftnA2 were the only down-‐regulated genes in R2866 that are regulated by Fur. The disparate down-‐regulation of Fur-‐associated genes in Rd and R2866 possibly represents their varied response to hydrogen peroxide. This is
supported by the fact that most of up-‐regulated Fur-‐regulated genes were only present in the R2866 strain, as described earlier.
The only gene locus that was down-‐regulated over 3-‐fold during oxidative stress in R2866 contained genes encoding proteins with putative roles in carbohydrate processing. The most highly down-‐regulated gene, Hgd, encoded a putative 2-‐(hydroxymethyl)glutarate dehydrogenase, which is involved in nicotinate fermentation in Eubacterium barkeri (Reitz et al., 2008). This gene is part of the same family of β-‐hydroxyacid dehydrogenases as gnd, which was up-‐ regulated in both strains. It is probable that the predicted annotation of this whole locus is wrong. Further investigation is required to determine the real function of this locus and its role in oxidative stress.