Lack of interchangeability of Hfq-like proteins

Hfq is an RNA-binding protein that participates in the regulatory activity of small non-coding RNAs (sRNAs) in many species of bacteria. Hfq protein was first crystallized from Staphylococcus aureus and this crystal structure constitutes a hallmark for bacterial Sm-like proteins. Paradoxically, however, the functional relevance/role of S. aureus Hfq (Hfq_SA) remains uncertain, as growing evidence suggests that the hfq_SA gene is expressed at very low levels or unexpressed in many S. aureus strains. To gather further insight, in the present work we exchanged the structural portion of the hfq gene of Salmonella enterica serovar Typhimurium (hfq_STM) with hfq_SA and analyzed the effects of the replacement on various Hfq-related phenotypes. Our results show that the replacement strain – in spite of expressing Hfq_SA at levels comparable to Hfq_STM in wild-type Salmonella – behaves as an hfq null mutant in three discrete small RNA-mediated regulatory responses. These defects correlate with an abrupt reduction in the intracellular concentration of sRNAs, as observed in an hfq null mutant. Failure of Hfq_SA to protect Salmonella sRNAs from degradation suggests that Hfq_Sa does not bind to these sRNAs. A parallel study with the Borrelia burgdorferi hfq gene (hfq_BB) gave essentially identical results: when made from a single copy chromosomal gene, Hfq_BB fails to substitute for Hfq_STM in sRNA-mediated regulation.     

Hfq regulates the expression of the thermostable direct hemolysin gene in <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Background  

The hfq gene is conserved in a wide variety of bacteria and Hfq is involved in many cellular functions such as stress responses and the regulation of gene expression. It has also been reported that Hfq is involved in bacterial pathogenicity. However, it is not clear whether Hfq regulates virulence in Vibrio parahaemolyticus. To evaluate this, we investigated the effect of Hfq on the expression of virulence-associated genes including thermostable direct hemolysin (TDH), which is considered to be an important virulence factor in V. parahaemolyticus, using an hfq deletion mutant.     

Hfq Is a Global Regulator That Controls the Pathogenicity of Staphylococcus aureus

The Hfq protein is reported to be an RNA chaperone, which is involved in the stress response and the virulence of several pathogens. In E. coli, Hfq can mediate the interaction between some sRNAs and their target mRNAs. But it is controversial whether Hfq plays an important role in S. aureus. In this study, we found that the deletion of hfq gene in S. aureus 8325-4 can increase the surface carotenoid pigments. The hfq mutant was more resistant to oxidative stress but the pathogenicity of the mutant was reduced. We reveal that the Hfq protein can be detected only in some S. aureus strains. Using microarray and qRT-PCR, we identified 116 genes in the hfq mutant which had differential expression from the wild type, most of which are related to the phenotype and virulence of S. aureus. Among the 116 genes, 49 mRNAs can specifically bind Hfq protein, which indicates that Hfq also acts as an RNA binding protein in S. aureus. Our data suggest that Hfq protein of S. aureus is a multifunctional regulator involved in stress and virulence.     

The RNA Chaperone Hfq Is Involved in Stress Tolerance and Virulence in Uropathogenic Proteus mirabilis

Hfq is a bacterial RNA chaperone involved in the riboregulation of diverse genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Proteus mirabilis to effectively colonize the bladder and kidneys in a murine urinary tract infection (UTI) model and to establish burned wound infection of the rats. In this regard, we found the hfq mutant induced higher IL-8 and MIF levels of uroepithelial cells and displayed reduced intra-macrophage survival. The loss of hfq affected bacterial abilities to handle H₂O₂ and osmotic pressures and to grow at 50°C. Relative to wild-type, the hfq mutant had reduced motility, fewer flagella and less hemolysin expression and was less prone to form biofilm and to adhere to and invade uroepithelial cells. The MR/P fimbrial operon was almost switched to the off phase in the hfq mutant. In addition, we found the hfq mutant exhibited an altered outer membrane profile and had higher RpoE expression, which indicates the hfq mutant may encounter increased envelope stress. With the notion of envelope disturbance in the hfq mutant, we found increased membrane permeability and antibiotic susceptibilities in the hfq mutant. Finally, we showed that Hfq positively regulated the RpoS level and tolerance to H₂O₂ in the stationary phase seemed largely mediated through the Hfq-dependent RpoS expression. Together, our data indicate that Hfq plays a critical role in P. mirabilis to establish UTIs by modulating stress responses, surface structures and virulence factors. This study suggests Hfq may serve as a scaffold molecule for development of novel anti-P. mirabilis drugs and P. mirabilis hfq mutant is a vaccine candidate for preventing UTIs.     

RpoS Regulates Essential Virulence Factors Remaining to Be Identified in Borrelia burgdorferi

Background

Since the RpoN-RpoS regulatory network was revealed in the Lyme disease spirochete Borrelia burgdorferi a decade ago, both upstream and downstream of the pathway have been intensively investigated. While significant progress has been made into understanding of how the network is regulated, most notably, discovering a relationship of the network with Rrp2 and BosR, only three crucial virulence factors, including outer surface protein C (OspC) and decorin-binding proteins (Dbps) A and B, are associated with the pathway. Moreover, for more than 10 years no single RpoS-controlled gene has been found to be critical for infection, raising a question about whether additional RpoS-dependent virulence factors remain to be identified.

Methodology/Principal Findings

The rpoS gene was deleted in B. burgdorferi; resulting mutants were modified to constitutively express all the known virulence factors, OspC, DbpA and DbpB. This genetic modification was unable to restore the rpoS mutant with infectivity.

Conclusions/Significance

The inability to restore the rpoS mutant with infectivity by simultaneously over-expressing all the three virulence factors allows us to conclude RpoS also regulates essential genes that remain to be identified in B. burgdorferi.     

Hfq variant with altered RNA binding functions

The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS–lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq–RNA interaction but has probably no effect on the folding and the oligomerization of the protein.     

Borrelia burgdorferi locus BB0795 encodes a BamA orthologue required for growth and efficient localization of outer membrane proteins

The outer membrane (OM) of the pathogenic diderm spirochete, Borrelia burgdorferi, contains integral β‐barrel outer membrane proteins (OMPs) in addition to its numerous outer surface lipoproteins. Very few OMPs have been identified in B. burgdorferi, and the protein machinery required for OMP assembly and OM localization is currently unknown. Essential OM BamA proteins have recently been characterized in Gram‐negative bacteria that are central components of an OM β‐barrel assembly machine and are required for proper localization and insertion of bacterial OMPs. In the present study, we characterized a putative B. burgdorferi BamA orthologue encoded by open reading frame bb0795. Structural model predictions and cellular localization data indicate that the B. burgdorferi BB0795 protein contains an N‐terminal periplasmic domain and a C‐terminal, surface‐exposed β‐barrel domain. Additionally, assays with an IPTG‐regulatable bb0795 mutant revealed that BB0795 is required for B. burgdorferi growth. Furthermore, depletion of BB0795 results in decreased amounts of detectable OMPs in the B. burgdorferi OM. Interestingly, a decrease in the levels of surface‐exposed lipoproteins was also observed in the mutant OMs. Collectively, our structural, cellular localization and functional data are consistent with the characteristics of other BamA proteins, indicating that BB0795 is a B. burgdorferi BamA orthologue.     

Characterization of 6S RNA in the Lyme disease spirochete

6S RNA binds to RNA polymerase and regulates gene expression, contributing to bacterial adaptation to environmental stresses. In this study, we examined the role of 6S RNA in murine infectivity and tick persistence of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. B. burgdorferi 6S RNA (Bb6S RNA) binds to RNA polymerase, is expressed independent of growth phase or nutrient stress in culture, and is processed by RNase Y. We found that rny (bb0504), the gene encoding RNase Y, is essential for B. burgdorferi growth, while ssrS, the gene encoding 6S RNA, is not essential, indicating a broader role for RNase Y activity in the spirochete. Bb6S RNA regulates expression of the ospC and dbpA genes encoding outer surface protein C and decorin binding protein A, respectively, which are lipoproteins important for host infection. The highest levels of Bb6S RNA are found when the spirochete resides in unfed nymphs. ssrS mutants lacking Bb6S RNA were compromised for infectivity by needle inoculation, but injected mice seroconverted, indicating an ability to activate the adaptive immune response. ssrS mutants were successfully acquired by larval ticks and persisted through fed nymphs. Bb6S RNA is one of the first regulatory RNAs identified in B. burgdorferi that controls the expression of lipoproteins involved in host infectivity.     

Molecular cloning and characterization of Borrelia burgdorferi rpoB

Borrelia burgdorferi rpoB, the gene encoding the β-subunit of RNA polymerase, has been cloned and sequenced. The full-length gene encodes a protein of 1154 amino acids with a calculated molecular mass of 129.8 kDa. The amino-acid sequence is 49% identical to the corresponding protein from Escherichia coli. B. burgdorferi rpoB is a component of a gene cluster, which includes rplJ, rplL and rpoC. A temperature-sensitive E. coli rpoB mutant could be complemented by introduction of the B. burgdorferi gene, indicating that the B. burgdorferi rpoB is expressed in E. coli and the β-subunit can be assembled into functional holoenzyme. The wild-type amino-acid sequence of the B. burgdorferi β-subunit is consistent with those of spontaneously arising rifampicin-resistant mutants of E. coli and Mycobacterium tuberculosis at certain critical residues. This suggests that the natural resistance of B. burgdorferi to rifampicin may be due to the primary amino-acid sequence of its β-subunit.     

Analysis of a Borrelia burgdorferi phosphodiesterase demonstrates a role for cyclic‐di‐guanosine monophosphate in motility and virulence

The genome of Borrelia burgdorferi encodes a set of genes putatively involved in cyclic‐dimeric guanosine monophosphate (cyclic‐di‐GMP) metabolism. Although BB0419 was shown to be a diguanylate cyclase, the extent to which bb0419 or any of the putative cyclic‐di‐GMP metabolizing genes impact B. burgdorferi motility and pathogenesis has not yet been reported. Here we identify and characterize a phosphodiesterase (BB0363). BB0363 specifically hydrolyzed cyclic‐di‐GMP with a K_m of 0.054 µM, confirming it is a functional cyclic‐di‐GMP phosphodiesterase. A targeted mutation in bb0363 was constructed using a newly developed promoterless antibiotic cassette that does not affect downstream gene expression. The mutant cells exhibited an altered swimming pattern, indicating a function for cyclic‐di‐GMP in regulating B. burgdorferi motility. Furthermore, the bb0363 mutant cells were not infectious in mice, demonstrating an important role for cyclic‐di‐GMP in B. burgdorferi infection. The mutant cells were able to survive within Ixodes scapularis ticks after a blood meal from naïve mice; however, ticks infected with the mutant cells were not able to infect naïve mice. Both motility and infection phenotypes were restored upon genetic complementation. These results reveal an important connection between cyclic‐di‐GMP, B. burgdorferi motility and Lyme disease pathogenesis. A mechanism by which cyclic‐di‐GMP influences motility and infection is proposed.     

Molecular characterization of a flagellar/chemotaxis operon in the spirochete Borrelia burgdorferi

A chemotaxis gene cluster from Borrelia burgdorferi, the spirochete that causes Lyme disease, was cloned, sequenced, and analyzed. This cluster contained three chemotaxis gene homologs (cheA, cheW and cheY) and an open reading frame we identified as cheX. Although the major functional domains for B. burgdorferi CheW and CheY were well conserved, the size of cheW was significantly different from the homolog of other bacteria. Phylogenetic analysis of CheY indicated that B. burgdorferi constitutes a distinct branch with Treponema pallidum and is closely associated with Archea and Gram-positive bacteria. RT-PCR analysis indicated that the chemotaxis genes and the upstream flagellar gene flaA constitute an operon. Western blot analysis using antibody to Escherichia coli CheA resulted in two reactive proteins in the cell lysates of B. burgdorferi that is consistent with two cheA homologs being present in this organism. The results taken together suggest both similarities and differences in the chemotaxis apparatus of B. burgdorferi compared to those of other bacteria.     

Turn-over of the small non-coding RNA RprA in E. coli is influenced by osmolarity

The sRNA RprA is known to activate rpoS translation in E. coli in an osmolarity-dependent manner. We asked whether RprA stability contributes to osmolarity-dependent regulation and how the RNA binding protein Hfq and the major E. coli endonucleases contribute to this turn-over. The study reveals that osmolarity-dependent turn-over of RprA indeed contributes to its osmolarity-dependent abundance. RprA is stabilized by the RNA chaperone Hfq and in absence of Hfq its turn-over is no longer osmolarity-dependent. The stability of the RprA target mRNA rpoS shows a lower extent of osmolarity dependence, which differs from the profile observed for RprA. Thus, the effect of sucrose is specific for individual RNAs. We can attribute a role of the endoribonuclease RNase E in turn-over of RprA and an indirect effect of the endoribonuclease III in vivo. In addition, RprA is stabilized by the presence of rpoS suggesting that hybrid formation with its target may protect it against ribonucleases. In vitro RprA is cleaved by the RNase E containing degradosome and by RNase III and rpoS interferes with RNase III cleavage. We also show that temperature affects the stabilities of the sRNAs binding to rpoS and of rpoS mRNA itself differentially and that higher stability of DsrA with decreasing temperature may contribute to its high abundance at lower temperatures. This study demonstrates that environmental parameters can affect the stability of sRNAs and consequently their abundance.     

Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003

Background

The RNA-binding protein Hfq is involved in stress and virulence of several pathogens, probably due to its role as mediator in small RNA (sRNA)-mRNA interactions. In this study, we investigate the function of Hfq in the Gram-positive pathogen Staphylococcus aureus, by constructing hfq null mutant derivatives.

Results

We report that unexpectedly, in S. aureus, Hfq does not seem to play a crucial role in stress response, RNAIII or spa mRNA quantity and exoprotein expression, as tested in three virulent genetic backgrounds. Moreover, a global analysis of the RN6390 hfq mutant, which tests ~ 2000 phenotypes, supports our results concerning the non-implication of Hfq in stress response, and shows that Hfq is also not involved in resistance to several chemical agents and antibiotics and does not seem to be implicated in metabolic pathways.

Conclusion

Our data suggest that although sRNA-mRNA interactions in S. aureus are decisive for gene expression regulation, they do not require the RNA-chaperone protein Hfq. These interactions possibly require an RNA-chaperone protein other than Hfq, which remains to be found.