In an attempt to identify meningococcal genes selectively up- or down-regulated in the intracellular environment of infected host cells we used a previously published RT-PCR-DD strategy that relies on the presence of highly and moderately repetitive transcribed DNA sequences in the meningococcal genome 8. Oligonucleotides designed on the basis of the DUS (DUS-IN and DUS-OUT) or the 26L, 27L nemis sequences (26L-IN, 26L-OUT, 27L-IN, 27L-OUT) were used as primers in reverse-transcriptase (RT) assays to prepare cDNAs from intracellular meningococci (strain B1940) recovered from saponin-lysed HeLa cells after 7 h of infection or control bacteria grown in DMEM without HeLa cells as previously described 7. After 7 h of infection bacterial recovery from infected cells reached the highest values 7. We decided to use HeLa cells because studies with these cells have contributed to our understanding of the molecular mechanisms underlying the infectious cycle of N. meningitidis 151617181920. The analysis of the meningococcal transcriptome during the early stages of an infection has been previously performed using HeLa cells 21.

The cDNAs from intracellular meningococci were then amplified by PCR using the corresponding oligonucleotides and a mixture of random hexamers as primers, and the PCRs were analyzed by polyacrylamide gel electrophoresis. By this approach, several bands corresponding to either up-regulated (a, c, d, e, f, g, l, m, n, o) or down-regulated genes (b, h, i) were detected in the intracellular bacteria (Fig. 1A). The bands a, c, f, g, l and n were then excised from the gels, the corresponding cDNAs were cloned and subjected to nucleotide sequences analysis. Three clones were sequenced form each band. The bands d-, e-, m-, and o-associated cDNAs, when subjected to cloning, each gave rise to more than one clone, and therefore they were not further analyzed in this study. Bands corresponding to down-regulated genes were not examined.

The nucleotide sequence analysis demonstrated that the band a- and f-associated cDNA corresponded to genes that have been shown to be induced in the intracellular environment and have been also implicated in meningococcal pathogenesis (Fig. 1C). The band a-associated cDNA corresponded to NMB1964, a gene of the GdhR-regulated gltT operon coding for the L-glutamate ABC transporter that is critical for meningococcal adaptation in the low-sodium intracellular environment 8. The band f corresponded to NMB0082 coding for LipA, a protein required for proper translocation and surface expression of the lipidated (α2→8)-linked polysialic acid capsule polymer 722. The band g-associated cDNA sequence fell within NMB1780-NMB1779 (hrpB-hrpA) operon coding for a two-partner secretion (TPS) system that has been recently shown to contribute to adhesion of un-encapsulated bacteria to epithelial cells 23. The bands c-, l- and n-associated cDNAs corresponded to genes whose role in meningococcal pathogenesis has never been reported so far, including NMB0551 (band c sequence) encoding PriA, the linked NMB0549 and NMB0550 (band l sequence) coding, respectively, for a thiol-disulfide oxidoreductase (DsbC) and the ATP-binding protein of an unknown ABC-type transporter (YbjZ), and NMB0954 (band n sequence) encoding GltA, the putative meningococcal citrate synthase (Fig. 1C). Fig. 1C also shows the genes surrounding priA (NMB0551). Upstream from NMB0549 (ybjZ) a gene coding for an AcrA/AcrE family protein (NMB0548) is located; downstream to priA and in the opposite direction a gene coding for a protein of unknown function (NMB0552) and that for a putative transposase (NMB0553) map.

The role of PriA in meningococcal pathogenesis was explored in more detail using the HeLa infection model. It is noteworthy that over-expression of priA was also observed in an independent screening by limited transcriptional analysis (Fig. 1B) that was useful to demonstrate up-regulation of lipA in a previous work 7. This screening was performed using a partial Sau3AI-restricted genomic library from B1940. The library was screened by Southern blot using cDNA probes derived from intracellular bacteria after 8 h of infection of HeLa cells or control bacteria grown for 8 h in cell culture medium. The screening led to isolation of a plasmid harboring two Sau3AI fragments, 983 and 1123 bp-long (marked by asterisks in Fig. 1B and 1C), spanning almost the entire priA gene.

Nevertheless, in order to confirm the results of the RT-PCR-DD analysis, slot blot and RT real-time PCR experiments were performed (Fig. 2). Semi-quantitative analysis by slot blot experiments demonstrated that the priA-specific RNA was about three-fold more abundant in meningococci recovered from saponin-lysed HeLa cells than in control bacteria grown in DMEM without HeLa cells (Fig. 2A–B). This result was confirmed by RT real-time PCR demonstrating an about four-fold increase in the amount of priA transcripts in intracellular meningococci (Fig. 2C). It should be noted that RT-PCR-DD, limited transcriptional analysis and RT real-time PCR were performed on RNAs from different sets of infection experiments thus enhancing the argument for a role of this gene in the intracellular environment.

To gain functional information about the role of priA in N. meningitidis, we decided to inactivate it insertionally by transformation with pDEXpriA (Fig. 3A and Materials and Methods). Southern blot analysis confirmed the insertion of this suicide plasmid into the priA coding region by a single cross-over event (Fig. 3B). As a result of the recombination event, two HinfI DNA fragments of the expected sizes (4019 bp and 852 bp) were detected by priA-specific probe in the transformed strains B1940ΩpriA (Fig. 3B, lanes 1–3) in place of the 1568 bp HinfI fragment of the parental strain B1940 (Fig. 3B, lane 4). In order to be sure that priA inactivation was causal to all of the phenotypes observed below, a functional copy of priA was introduced into leuS-Ψdam region of B1940ΩpriA by using the integrative plasmid pACpriA. Successful gene delivery was demonstrated by Southern blot analysis (Fig. 3C). In two transformants (B1940ΩpriA/priA+) the priA-specific probe detected the presence of both the 1568 bp HinfI fragment (typical of the wild type priA) and the 4019 bp and 852 bp HinfI fragments (typical of the inactivated priA) (Fig. 3C, lanes 3 and 4).

Phenotypic analysis demonstrated that priA inactivation resulted in a growth defect. Indeed, under aerobic conditions, B1940ΩpriA strain failed to grow in diluted inocula (= or <10⁶ CFU ml^-1), but when it was inoculated at higher densities (= or >10⁷ CFU ml^-1) it grew at rates similar to those of the parental strain (Fig. 4A). However, in high-density inocula viability of B1940ΩpriA as determined by CFU was apparently reduced when compared to that of B1940. In the experiment shown in Fig. 4C, left panel, B1940, B1940ΩpriA and B1940ΩpriA/priA+ were grown aerobically in liquid GC medium to late logarithmic phase (1.0 OD_{600 nm}). Then, serial dilutions of the cultures were plated on GC agar, incubated aerobically in the presence of 5% CO₂, and the number of CFU determined after 24 h. Results demonstrated small colony size (Fig. 4C) and reduced colony number (to about 34%) of the priA-defective strain compared to the parental one (Fig. 4B). This phenotype was much more severe when GC agar plates containing nitrite were incubated under either aerobic (not shown) or oxygen-limiting conditions (Fig. 4C, right panel). Under both these conditions the colony number of B1940ΩpriA was reduced to about 15% when compared to that of B1940 under the same conditions (Fig. 4B). Statistical analysis with the Student's T-test confirmed that the growth defects in the presence of nitrite or in oxygen limited conditions in the presence of nitrite were more severe than under aerobic conditions without nitrite (p value < 0.05). The growth defect of B1940ΩpriA was not observed during middle logarithmic phase (data not shown), and could be completely restored by complementation with pACpriA in B1940ΩpriA/priA+ (Fig. 4B and 4C).

We next investigated the mechanism underlying the growth defect of B1940ΩpriA. As there is evidence that in E. coli priA inactivation results in defective cell division that generates long filaments 24, we used scanning electron microscopy (SEM) to analyze morphology B1940ΩpriA. Indeed, the growth defect of this strain as determined by CFU method might have been caused by a cell division defect that in meningococci, which divide in alternating planes generates clumps. However, SEM analysis did not reveal any difference between the wild type and the priA-defective strain grown to late logarithmic phase (1.0 OD_{600 nm}) (Fig. 5).

To confirm the hypothesis that the growth impairment of B1940ΩpriA might be due to reduced viability, the Live/Dead staining assay was carried out. This assay employs two fluorescent nucleic acid stains, SYTO9 and propidium iodide, which differ in their ability to penetrate healthy bacterial cells. When used alone, the green-fluorescent SYTO 9 stain labels both live and dead bacteria. In contrast, the red-fluorescent propidium iodide stain penetrates only bacteria with damaged membranes, reducing SYTO 9 fluorescence when both dyes are present. Thus, live bacteria fluoresce green, while dead bacteria fluoresce red (Fig. 6A). Using this system we did not observe any significant changes in viability between B1940 and B1940ΩpriA when stain was carried out with bacteria grown in GC medium to middle logarithmic phase (0.5 OD_{600 nm}). Indeed, the percentage of nonviable cells was approximately 10% for both strains. In contrast, B1940ΩpriA exhibited marked loss of viability during late logarithmic phase (1.0 OD_{600 nm}) when the percentage of nonviable cells approximated 35% (Fig. 6B). The defect in viability was completely restored by complementation (Fig. 6A and 6B). The Live/Dead staining assay also confirmed the absence of any cell division defect in B1940ΩpriA (Fig. 6A).

Assessment of viability after exposure to hydrogen peroxide (Fig. 7A) or the nitric oxide generator sodium nitroprusside (Fig. 7B) also demonstrated that B1940ΩpriA was much more sensitive to oxidative and nitrosative injuries when compared to the parental strain. The phenotype could be complemented back to wild type levels in B1940ΩpriA/priA+.

B1940, B1940ΩpriA and B1940ΩpriA/priA+ were then used in cell invasion and intracellular persistence assays. In these experiments, high density-inocula of meningococci were used and infections were initiated by using comparable numbers of viable bacteria for both strains as determined by both CFU method and Live/Dead staining. After allowing meningococci to invade HeLa cells for 1 h, intracellular viability was assessed immediately after gentamicin (time 0 h) treatment and 3, 5 and 7 h post-gentamicin treatment (Fig . 8). Results demonstrated the absence of statistically relevant differences in the ability to invade the HeLa cells by the three strains (Fig. 8, time 0 h). At 3 h post-gentamicin treatment the number of recoverable wild type, mutant and complemented bacteria dropped about ten-fold. Normally, at this time, a decrease occurs in intracellular viable bacteria, which is also characteristic of gonococcal invasion 782526. Between 3 and 7 h B1940 and B1940ΩpriA/priA+ underwent fast duplication and the number of viable bacteria increased more than 50-fold. In particular, intracellular duplication was extremely fast during the first 2 h after gentamicin treatment (time 3–5 h) with an apparent generation time of about 24 min and slower during the last 2 h (time 5–7) consistent with previous findings 78. In contrast, between 3 and 7 h the number of viable B1940ΩpriA bacteria increased less than 10-fold with an apparent generation time of 96 min during the first 2 h after gentamicin treatment (time 3–5 h). It should be noted that intracellular generation times are only apparent, as they reflect the sum of intracellular growth, intracellular death and, eventually, bacterial egression from HeLa cells and re-invasion. Therefore different processes may be affected in this mutant.

To further investigate the origin of the growth/survival defect of B1940ΩpriA inside HeLa cells we used immunofluorescence microscopy. Intriguingly, we noted that number of intracellular priA-defective bacteria 7 h after infection was about the same as those of wild type and complemented bacteria (Fig. 9 and data not shown). This seemed to be in contrast with the data obtained with the CFU method showing a marked decrease in the number of intracellular priA-defective bacteria 7 h after infection (Fig. 8). However, in contrast to CFU method, immunofluorescence analysis detects even dead bacteria, and this means that most priA-defective meningococci inside HeLa cells were actually not viable. The evidence that, at variance with wild type and complemented bacteria, most priA mutant bacteria co-localized with the lysosomal associated membrane protein 1 (LAMP1) strongly supported this hypothesis. This finding is noteworthy because live meningococci have been shown to use IgA protease to cleave LAMP1 protein 27, accounting for the lack of co-localization of wild type and complemented bacteria with this marker. On the basis of this evidence we concluded that the growth phenotype of the priA-defective mutant inside HeLa cells was mostly due to reduced viability than to reduced growth rate.

N. meningitidis serogroup B strain B1940 was used in this study. The origin and genotype of this strain have been previously reported 42. Meningococci were cultured either aerobically in GC broth or agar with 1% Polyvitox, at 37°C in a 5% CO₂ incubator or under oxygen-limiting conditions in GC agar containing 5 mM sodium nitrite with 1% Polyvitox, at 37°C in an anaerobic jar with an anaerobic atmosphere generator (GENbox anaer, Biomerieux) and an indicator (Anaer indicator, Biomerieux).

E. coli strain DH5α [F^-Φ80d lacZΔM15 endA1 recA1 hsdR17 supE44 thi-1 λ^- gyrA96 Δ(lacZYA-argF) U169] was used in cloning procedures. This strain was grown in Luria Bertani (LB) medium. To allow plasmid selection, the LB medium was supplemented with ampicillin (50 μg ml^-1).

For standard invasion and intracellular viability assays, HeLa cells (ATCC Number CCL-2) were used. Several experiments were also repeated with HEp2 (ATCC Number CCL-23) and Chang conjunctiva cells (ATTC Number CCL-20.2). It should be noted, however, that, as a result of a well known contamination event, HEp2 (thought to be derived from an epidermoid carcinoma of the larinx) and Chang conjunctiva (thought to be derived from normal conjunctiva) cells present HeLa markers and have been established via HeLa cells contamination, as stated by ATCC http://www.atcc.org. These cells were grown at 37°C in a 5% CO₂ incubator in DMEM supplemented with inactivated 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (50 U ml^-1) and streptomycin (50 μg ml^-1).

N. meningitidis invasion assays were performed as previously described 87. In brief, HeLa (or HEp2 or Chang conjunctiva) cells were infected at a multiplicity of infection (MOI) of 50 for 1 h. To start the infection bacteria were centrifuged (60 × g) onto cells. Cells were washed twice with phosphate-buffered saline (PBS) to eliminate the majority of extracellular bacteria and exposed to gentamicin to kill remaining extracellular bacteria. Cells were then washed extensively with PBS to remove gentamicin and dead extracellular bacteria, and then lysed with saponin. Gentamicin treatment was performed at 100 μg ml^-1, a concentration 10-fold above the minimal inhibitory concentration (MIC) for 30 min. When required, cells were re-incubated in a fresh culture medium for various time intervals after gentamicin treatment. For quantification, bacteria released by saponin from HeLa cells (intracellular) were plated and colony-forming units (CFU) were counted the day after.

High-molecular-weight genomic DNA from N. meningitidis strains was prepared as described previously 43. Oligonucleotides used in this study as primers in PCRs are listed in Table 1. Oligonucleotide synthesis was performed as a service by MWG-Biotech AG Oligo Production. The amplification reactions generally consisted of 35 cycles including 45 s of denaturation at 94°C, 45 s of annealing at 65°C, and 45 s of extension at 72°C. They were carried out in a Perkin-Elmer Cetus DNA Thermal Cycler 2400. DNA from strain B1940 was used as a template. Southern blot hybridizations were carried out according to standard protocols 44. ³²P labeling of the DNA fragments was performed by random priming using the Klenow fragment of the E. coli DNA polymerase I and [α-³²P]dATP and [α-³²P]dGTP (3,000 Ci mmol^-1) 44. DNA sequencing was performed as a service by the MWG Biotech Custom Sequencing Service. Processing of the DNA sequences was performed with the software GeneJockey Sequence Processor (published and distributed by Biosoft). Deduced amino acid sequence similarity searching was performed with the BLAST program using the Conserved Domain Database available at the NCBI.

The Neisseria-E. coli shuttle vectors pDEX and pACNL1 have been previously described 945. pACNL1 is a pACYC184 derivative harboring a 1480 bp Sau3AI fragment, spanning the meningococcal leuS-Ψdam region in the BamHI site. To construct pDEXpriA, the DNA corresponding to the central segment of the ORF NMB0551 was amplified using the primers NMB0551-1 and NMB0551-2, and the BamHI-restricted 851 bp PCR product was cloned into the BamHI site of pDEX. NMB0551 (priA) was inactivated in B1940 by single cross-over event using pDEΔpriA originating the strain B1940ΩpriA. Transformations were performed by using 0.1 to 1 μg of plasmid DNA. Transformants were selected on GC agar medium supplemented with erythromycin (7 μg ml^-1). Successful gene inactivation was demonstrated by Southern blot hybridization. NMB0551-specific probe was obtained by ³²P labeling of BamHI-restricted PCR fragment. The size of this fragment was 851 bp (primers NMB0551-1 and NMB0551-2). B1940ΩpriA was complemented by transformation with the integrative plasmid pACpriA. To construct this plasmid, the priA gene (with flanking regulatory elements) was amplified from strain B1940 using the primers NMB0551-3 and NMB0551-4 (Table 1). The XbaI/HindIII-restricted 2565 bp PCR product was cloned into the XbaI/HindIII sites of pACNL1. Transformants were selected on GC agar medium supplemented with chloramphenicol (5 μg ml^-1).

Total RNAs were extracted from intracellular bacteria during the infection of HeLa cells or control bacteria grown in culture medium as described 8.

The procedure for limited transcriptional analysis has been previously detailed 98. Briefly, a partial Sau3AI-restricted genomic library from the serogroup B strain B1940 was constructed. Individual clones were digested and a Southern blot analysis was performed using cDNA probes derived from intracellular bacteria after 8 h of infection of HeLa cells or control bacteria grown for 8 h in cell culture medium. This procedure detects mRNA species present in the initial population at a frequency of at least 1 in 200.

The protocol for RT-PCR-DD analysis has been also reported 8. Briefly, total RNAs were extracted from intracellular bacteria during the infection of HeLa cells or control bacteria grown in cell culture medium, and were used as templates for first-strand cDNA synthesis in the presence of the primers DUS-IN, DUS-OUT, 26L-IN plus 27L-IN, 26L-OUT plus 27L-OUT, RS3-IN, RS3-OUT listed in Table 1. Then second-strand cDNAs synthesis was carried out using the corresponding oligonucleotides and a mixture of random hexamers as primers, and the PCR products were analyzed by polyacrylamide gel electrophoresis. The gel was stained by the silver staining method 44. The slices of gel containing the differentially expressed transcripts were cut with a sharp scalpel, and the DNA fragments were allowed to diffuse into 100 μl of sterile water at 37°C for 4 h under stirring. The eluted DNA was ethanol precipitated using glycogen as a carrier. Then, it was used as a template in a PCR with the corresponding primers. The positive amplicons were cloned in a pGEM easy vector (Promega) and sequenced.

RT-PCR slot blot analysis of meningococcal priA-specific transcripts during infection of HeLa cells was performed as previously described 8. To prepare gene-specific cDNA probes, the RNAs were subjected to reverse transcription using the oligonucleotides NMB0551-2 or 16Suniv-2 (loading control) as primers. The labeling procedure consisted of a PCR, where 2 μl of cDNA was used as template in a 50 μl mixtures containing 0.2 mM of each dCTP and dTTP, 2.5 μM of each dATP and dGTP, 0.12 μM [α-³²P]ATP (3000 Ci mmol^-1) and [α-³²P]GTP (3000 Ci mmol^-1), 0.2 μM forward NMB0551-1 or 16Suniv-1 primers, and 0.05 U μl^-1 of Taq polymerase (Perkin Elmer S.p.A). The amplification reaction consisted of 20–25 cycles including 45 sec of denaturation at 94°C, 45 sec of annealing at 55°C and 45 sec of extension at 72°C. The number of cycles was critical to operate in the linear range of the PCR, and it was determined in preliminary experiments. The ³²P-labeled cDNA probes were hybridized to different amounts (0.05 to 100 ng) of denatured NMB0551 (priA)- or 16S rRNA gene-specific DNA fragments generated by PCR with the corresponding primer pairs NMB0551-1/NMB0551-2, and 16Suniv-1/16Suniv-2, and fixed onto positively charged Hybond-N+ nylon membranes. The hybridization reactions were carried out in Church buffer 44 at 63°C. Semi-quantitative analysis was performed by directly counting the radioactivity bands by a PhosphoImager SI (Molecular Dynamics, Inc., Sunnyvale, CA).

Semi-quantitative analysis of the priA-specific transcript, normalized to 16S rRNA, was also performed by real-time RT-PCR. Total RNAs (1 μg) from either intracellular or control bacteria grown in cell culture medium were reverse-transcribed by using random hexamer (2.5 μM) with Superscript RT (Invitrogen). About 0.1–1% of each RT reaction was used to run real-time PCR on a SmartCycler System (Cepheid) with SYBR^® Green JumpStart Taq ReadyMix (Sigma-Aldrich) and the primer pairs 16Suniv-1/16S-r (specific for 16S rRNA) and NMB0551-f/NMB0551-r (specific for priA). PCR products were 185 bp for 16Suniv-1/16S-r, and 143 bp for NMB0551-f/NMB0551-r. Real-time PCR samples were run in triplicate. The real-time PCR conditions were: 30 sec at 94°C, 30 sec at 55°C, 30 sec at 72°C for 35 cycles; detection of PCR products was performed at 83°C.

The viability of meningococcal strains was determined by using the Live/Dead BacLight bacterial viability kit (Molecular Probe). To this purpose, meningococci were grown to either middle (0.5 OD_{600 nm}) or late (1.0 OD_{600 nm}) logarithmic phase in 50 ml of GC medium with 1% Polyvitox as described above. Then 1 ml of bacterial suspension was collected by low-speed centrifugation. Meningococci were washed, re-suspended in 1 ml 0.9% NaCl and mixed with an equal volume of 2X working solution of Live/Dead BacLight containing a 1:1 mixture of SYTO9 and propidium iodide. After 15 min dark incubation, 5 μl of mounted specimens were viewed with a Nikon Optiphot-2 microscope with an episcopic-fluorescence attachment (EFD-3, Nikon).

B1940, B1940ΩpriA and B1940ΩpriA/priA+ were grown to middle logarithmic phase (0.5 OD_{600 nm} corresponding to about 1 × 10⁹ CFU). Then, 5 ml aliquots ("input" CFU = 5 × 10⁹ CFU for each strains) were placed into 15-ml conical tubes. H₂O₂ was added to the tubes at final concentrations of 0, 0.5, or 1 mM, and tubes were incubated with shaking for 20 min at 37°C. Cultures were immediately centrifuged and pellets were re-suspended into 5 ml GC broth. These re-suspensions were serially diluted into GC broth and spotted onto GC agar. The plates were incubated at 37°C in a 5% CO₂ incubator and the colonies were counted after 24 h of growth. For the sodium nitroprusside sensitivity assay, dilutions of freshly grown liquid cultures of N. meningitidis to middle logarithmic phase (0.5 OD_{600 nm} corresponding to about 1 × 10⁹ CFU) were immediately spread onto GC agar plates containing 1% Polyvitox and the nitric oxide generator. Plates were incubated in an atmosphere of 5% CO₂ at 37°C.

Immunofluorescence analysis was performed as previously described to distinguish between extracellular and intracellular bacteria 7. In brief, after infection, cells were washed once with PBS, and fixed with 3% paraformaldehyde. For staining of extracellular bacteria, incubation with primary antibodies was carried out for 20 min at room temperature. After washes in PBS, cells were incubated with secondary antibodies for 20 min in the dark, at room temperature. Subsequently, cells were permeabilized with saponin, incubated with primary antibodies to stain bacteria or intracellular markers and then incubated with secondary antibodies. Rabbit polyclonal anti-N. meningitidis antibody was from ViroStat while monoclonal H4A3 anti-LAMP1 was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa. Primary and secondary antibodies were used at a 1:500 dilution. Mounted coverslips were examined using a Nikon Optiphot-2 microscope with an episcopic-fluorescence attachment (EFD-3, Nikon). Image processing was carried out with Adobe Photoshop version 7.0.

Meningococcal strains B1940 and B1940ΩpriA were grown to late (1.0 OD_{600 nm}) logarithmic phase in 50 ml of GC medium with 1% Polyvitox as described above. Then 1 ml of bacterial suspension was collected by centrifugation. For SEM observations, meningococci were fixed with 1% glutaraldehyde, washed three times with distilled water by centrifugation, dehydrated in a graded alcohol series and critical-point dried. The sample was then mounted on Aluminum stubs, sputter-coated with gold and examined at an accelerating voltage of 20 kV with a Jeol 6060LV Scanning Electron microscope.