Plasmodium sporozoites are abruptly subjected to the environment of the warm-blooded host often after extended periods of quiescence in the insect’s salivary gland lumen. In the mammalian host, the inoculated sporozoites are likely to remain extracellular for a few hours before infecting the hepatocytes, the only cell type where they can develop to maturity. We hypothesized that during this period, sporozoites are activated to a state of readiness for hepatocyte invasion. Parasite material corresponding to this transition period suitable for molecular investigations would be very difficult to obtain in vivo, especially if P. falciparum sporozoites are to be investigated.
We explored the influence of temperature and host cell contact, the two host environmental factors amenable to investigation in vitro
, on the infectivity of P. falciparum
sporozoite to hepatocytes 
. We demonstrated that a temperature shift to 37°C is required to make salivary gland sporozoites infective to hepatocytes, though infectivity is lost within 30 minutes when only the sporozoites are incubated at this temperature. We showed that the loss in the P. falciparum
sporozoites' infectivity can be prevented when incubation at 37°C is made in the presence of either of two types of human cells, skin keratinocytes or primary hepatocytes ().
Recently, in vivo
studies in rodents showed that the majority of sporozoites are deposited in the skin 
and migrate away from the site of inoculation over the next few hours 
. One could speculate that residence in the mammalian host for a few hours is needed to bring the majority of the salivary sporozoites from a state of quiescence to that of hepatocyte-invasion readiness. Our observations performed in vitro
with a human skin cell line or with human primary hepatocytes are consistent with this notion. The role of higher temperature (37°C) in optimal sporozoite infectivity might be simply explained in terms of the metabolic activation required to power motility. However, our results and previous studies indicate that this temperature shift has more wide-ranging consequences. First, sporozoites motility per se
occurs without a temperature rise in the mosquito since the sporozoites released from the oocysts into the haemolymph migrate to and invade salivary gland sporozoites, albeit the mechanisms and speed of migration might differ from those in the mammalian host. Second, it was shown that a shift to 37°C activated exocytosis of P. falciparum
sporozoite micronemes, a phenomenon associated with productive infection of hepatocytes, and this was enhanced when parasites were incubated with hepatocytes 
. Third, a mere shift to 37°C, in the presence of serum, was sufficient to induce the transformation of P. berghei
sporozoites into forms morphologically indistinguishable from early EEF 
. Nonetheless, the importance of host cell contact was clearly demonstrated by the ability of hepatocytes to preserve sporozoite infectivity during lengthy incubation at 37°C ().
We considered that the infectious sporozoites obtained after incubation in the co-cultures were physiologically similar to those found in vivo a few hours after the infectious mosquito bite. Thus, it was possible to obtain large numbers of viable and infectious sporozoites after incubation at 37°C in the presence of hepatocytes, so as to conduct whole transcriptome profiling analyses that would identify the modifications in steady-state levels of transcripts induced by the insect-to-mammalian host transition.
It was clear that exposure of salivary gland sporozoites to 37°C in the presence of hepatocytes for merely one hour, triggered complex and genome-wide changes in transcript levels. This substantial gene modulation probably participated to prepare, or possibly to activate, the sporozoite for successful infection. Indeed, the mRNA levels for 611 genes were altered in activated P. falciparum sporozoites, and for most genes (532/611) there was up-regulation. The genes identified included those that encode proteins involved in parasite transcription, translation, signalisation pathways, transportation, metabolism and also invasion. These observations are consistent with the concept that sporozoites that are ready to invade the hepatocyte productively are functionally different from the salivary gland sporozoites inoculated by the mosquito.
To date, little data is available on gene expression in P. falciparum
pre-erythrocytic stages. Three genome wide expression data sets have become recently available for P. yoelii
pre-erythrocytic stages: a cDNA library from sporozoites transformed axenically into early EEF forms, where 652 unique transcripts were identified 
; a cDNA library of laser capture microdissected mature liver stages, where 623 unique transcripts were identified 
; and most recently the transcriptome of liver stages at three points during their development, where 1985 actively transcribed genes were identified 
. From a technical point of view, the data we present is most significantly comparable to that of Tarun et al.
since in both cases microarray analysis of the transcriptome was carried out, and changes in steady state levels were used as a criterion for gene identification. However, the data from Wang et al.
, is biologically more relevant to our data, since it is derived from sporozoites that had been incubated at 37°C. Of the 611 P. falciparum
genes identified in this study, 120 did not have orthologues in any of the three species that infect rodents according to orthology mapping data of Tarun et al.
2008 (see Figure S2
and Table S1
). Of the remaining 491 P. falciparum
genes with such orthologues, 321 were not represented in the sets identified in P. yoelii
transformed sporozoites by Wang et al.
2004, or in the liver stage parasites used by Tarun et al.
2008. These relatively low levels of overlap are probably due to the fact that the data sets were derived from different stages: maturing hepatic stage parasites 
or sporozoites incubated at 37°C for 24 hours in the absence of hepatocytes 
as compared to the sporozoites incubated for 1 hour in the presence of hepatocytes from which our data was derived.
Confirmation that the microarray data presented reliably identified up-regulated genes was obtained through independent RT-qPCR analysis of a subset of 21 up-regulated genes. The RT-qPCR analysis further afforded the opportunity to investigate the time course of up-regulation over two hours of activation, and to explore the relative contribution of temperature or host cell contact on the alterations in gene expression. Two distinct patterns of up-regulation were observed (). It was hypothesised that these patterns can provide an indication as to the likely role of the corresponding proteins. The modest transient up-regulation observed following incubation at 37°C in the presence of hepatocytes was characteristic for the selected genes known to be associated with invasive processes. In this case, it would appear that contact with hepatocytes alone accounted for the observed up-regulation (), since no up-regulation was observed when the sporozoites were incubated at 37°C without hepatocytes. For the group of genes that included those encoding proteins known or likely to be expressed during hepatic stage development, up-regulation increased continuously with the duration of incubation at 37°C in the presence of hepatocytes, and the levels reached tended to be high. In this case, the shift in temperature seemed to be the effective signal (), since similar changes in expression were observed irrespective of the contact with hepatocytes. At this stage, it is not possible to conclude that these increases translate into higher protein levels. Although global analysis of protein and RNA levels showed a positive correlation between mRNA and protein abundance, delays between mRNA and protein accumulation were noted for many genes 
. This issue can only be addressed by analysis of individual genes and their products.
Functional studies of two hypothetical proteins encoded by genes with the transient pattern of up-regulation, confirmed that they were likely to be implicated in host cell invasion. IFA staining patterns suggested that SIAP-1 and SIAP-2 were located at the surface of the sporozoite. Western blotting showed that they were released in the supernatant upon incubation at 37°C, and that SIAP-1 was cleaved after release. Further studies are required to determine whether cleavage is achieved using the same proteolytic machinery implicated in the shedding of TRAP and AMA-1 
. Cell invasion inhibition assays showed that both anti-SIAP-1 and anti-SIAP-2 blocked sporozoite migration and productive invasion of hepatocytes, further supporting surface localization. The fact that inhibition levels for cell traversal were similar to those afforded by anti-PfCSP antibodies, while those for hepatocyte invasion were lower (in particular for SIAP-2) suggest that SIAP-1 and SIAP-2 might have a more prominent role in sporozoite gliding than in hepatocyte invasion. Future studies would require inhibitory activities to be measured in terms of the quantity of specific antibodies rather than in terms of dilution to evaluate the potential of these 2 proteins as vaccine candidates. It should be noted that the P. yoelii
orthologue of SIAP-1 was detected in the proteome of the mature liver stages of this parasite 
, but not in that of P. berghei
parasites. It is not clear at present whether the IFA negative results obtained for P. falciparum
liver stage reflect the absence of SIAP-1, post-translational modifications that adversely affect antibody recognition, or species differences. The modest increases noted for transiently up-regulated genes is consistent with proteins associated with motility and/or invasion, because these proteins are likely to be already present in salivary gland sporozoites, as is the case for TRAP and aldolase that participate in the motor machinery powering gliding motility 
. At best, the transient up-regulation of invasion-related genes might serve to compensate for the loss of invasive proteins shed by the parasite during migration to its final target cell.
Characterization of the two hypothetical proteins encoded by genes with the continuous pattern of up-regulation showed that they were expressed throughout hepatic parasite development. The two corresponding genes had reached high expression levels as a result of activation. Detectable increases in LSAP-1 protein levels were noted as early as day 1 post-inoculation in transforming sporozoites, whereas LSAP-2 protein could only be detected from day 2 post inoculation. LSAP-1 was mainly found at the periphery of the intracellular hepatic parasite throughout its development, but not in blood stage parasites and possibly in minor quantities in salivary gland sporozoites. Thus, LSAP-1 might represent the second liver stage-specific protein to be identified in P. falciparum
, the other one being LSA-1 
. LSAP-2 was also mainly expressed at the periphery of the intracellular hepatic parasite, but it was additionally detectable in moderate quantities in blood stage parasite though not in salivary gland sporozoites. The presences of a PEXEL/VTS domain in LSAP-2 and of a signal peptide in LSAP-1 are consistent with location at the PVM and eventual export to the host cell cytoplasm, though the proteins were not detected in the cytoplasm of infected hepatocytes. Knock-out studies, such as those used for UIS3 
, UIS4 
and P36p 
will be required to ascribe a function for these protein or to establish whether they are essential for liver stage development. It is interesting to note that there were no identifiable orthologues for either gene in the rodent model species, as was the case for SIAP-2 and LSA-1, which suggests that they might interact with components specific to the primate hepatocyte.
In conclusion, whole genome transcriptome analysis of P. falciparum
sporozoite activated by relatively brief exposure to mammalian host-like conditions, revealed major changes in gene expression. A fifth of genes identified as being up-regulated in activated P. falciparum
sporozoites had no orthologues in the genomes of the Plasmodium
species used as experimental models in rodents. Some of the proteins encoded by these genes might have specifically evolved to optimise the interactions between the parasite and the human liver. The data presented provides a gateway for the identification of new P. falciparum
genes implicated in the processes of hepatocyte invasion and liver stage development, and as such some of these might prove valuable as potential vaccine targets. In a previous study, 16 P. falciparum
sporozoite proteins were identified as being highly antigenic based on stimulation of immune cells obtained from volunteers immunized with radiation-attenuated sporozoites 
. Five of these proteins including SIAP-1 were encoded by genes up-regulated in the activated sporozoite transcriptome (Table S1
). It is hoped that future studies might provide the basis for unravelling the biology of, and eventually elaborate therapies against, the few short-lived stages that occur between the bite of an infected mosquito and the release of merozoites, which constitute the obligatory steps without which the establishment of a Plasmodium
infection cannot occur.