Acid Phosphatases Do Not Contribute to the Pathogenesis of Type A Francisella tularensis

The intracellular pathogen Francisella tularensis is the causative agent of tularemia, a zoonosis that can affect humans with potentially lethal consequences. Essential to Francisella virulence is its ability to survive and proliferate within phagocytes through phagosomal escape and cytosolic replication. Francisella spp. encode a variety of acid phosphatases, whose roles in phagosomal escape and virulence have been documented yet remain controversial. Here we have examined in the highly virulent (type A) F. tularensis strain Schu S4 the pathogenic roles of three distinct acid phosphatases, AcpA, AcpB, and AcpC, that are most conserved between Francisella subspecies. Neither the deletion of acpA nor the combination of acpA, acpB, and acpC deletions affected the phagosomal escape or cytosolic growth of Schu S4 in murine and human macrophages, despite decreases in acid phosphatase activities by as much as 95%. Furthermore, none of these mutants were affected in their ability to cause lethality in mice upon intranasal inoculation. Hence, the acid phosphatases AcpA, AcpB, and AcpC do not contribute to intracellular pathogenesis and do not play a major role in the virulence of type A Francisella strains.The Gram-negative bacterium Francisella tularensis is a highly infectious, facultative intracellular pathogen that causes tularemia, a widespread zoonosis affecting humans. Human tularemia is a fulminant disease that can be contracted by exposure to as few as 10 bacteria, the pneumonic form of which can lead to mortality rates as high as 25% if untreated (35). Three subspecies of F. tularensis, Francisella tularensis subsp. tularensis (type A), Francisella tularensis subsp. holarctica (type B), and Francisella tularensis subsp. mediasiatica, are recognized, among which strains of the first two subspecies can cause tularemia in humans (15). While type B strains are geographically distributed all over the northern hemisphere, the highly virulent type A strains are restricted to North America and account for the most-severe cases of the disease. Francisella novicida, a species of low virulence in humans but high virulence in rodents, has been used extensively as a surrogate model of F. tularensis pathogenesis, based on the assumption that it uses conserved virulence mechanisms (4, 7, 8, 19, 23, 25-29, 31, 41-45, 47). As a facultative intracellular pathogen, F. tularensis is capable of infecting and proliferating in a variety of host cell types, including hepatocytes, epithelial cells, and mononuclear phagocytes (15). Macrophages constitute an important target for infection in vivo (21), and the pathogenesis of F. tularensis depends on the bacterium''s ability to survive and replicate within these host cells (15). Upon phagocytosis, Francisella ensures its effective survival and proliferation via rapid phagosomal escape followed by extensive replication in the cytosol (11, 14, 20, 42), thereby segregating itself from the degradative endosomal system and its associated bactericidal activities. Phagosomal escape is a tightly regulated process whose efficiency depends on conditions encountered within the early phagosome (12, 41), such as vacuolar acidification, although some controversy remains as to whether Francisella-containing phagosomes are significantly acidified prior to membrane disruption (13). Regardless of such discrepancies, phagosomal escape is an essential step in Francisella intracellular pathogenesis, since it is a prerequisite for cytosolic replication. Indeed, Francisella mutants that are defective in phagosomal escape do not grow intracellularly and are attenuated in vivo (6, 24, 43-45), and a belated phagosomal escape delays intracellular proliferation of the highly virulent type A strain Schu S4 (12).Much effort has focused on identifying bacterial factors that contribute to phagosomal escape. Several genes located within a 30-kb chromosomal locus known as the Francisella pathogenicity island (FPI) (31) are required for proper phagosomal escape of F. novicida (43, 44) and the attenuated F. tularensis subsp. holarctica live vaccine strain (LVS) (6, 24), since transposon insertions or targeted deletions in iglC, iglD, and pdpA affect the translocation of the mutants to the cytosol. Based on the homology of some FPI proteins with components of type VI secretion systems in other pathogens (30, 36), the FPI likely encodes a secretion apparatus that is required for phagosomal disruption. Yet a true understanding of FPI functions and the characterization of actual Francisella effectors of phagosomal escape are lacking. In addition to the FPI, Mohapatra et al. have recently reported for F. novicida that the acid phosphatases AcpA, AcpB, AcpC, and Hap are required for phagosomal escape and virulence in mice (27, 29). Acid phosphatases, which are ubiquitous in nature and hydrolyze phosphomonoesters at acidic pHs, have been associated with the survival of intracellular parasites within phagocytes through inhibition of the respiratory burst (1, 3, 9, 22, 37-40), suggesting that they act as virulence factors. In Francisella, a prominent role was established for AcpA, an unusual, respiratory-burst-inhibiting enzyme exemplifying a novel family of acid phosphatases (18, 37). AcpA accounts for most of the acid phosphatase and phospholipase activities in the outer membrane fraction of F. novicida (29). These reports assigned acid phosphatases a role in phagosomal escape yet contradicted a previous study by Baron et al., who concluded that AcpA was not required for the intracellular growth or virulence of F. novicida (4). While the acpA mutants were constructed differently in these studies, the acid phosphatase activity associated with AcpA was abolished in both situations. A proposed explanation for these conflicting results was that the truncated AcpA generated by Baron et al. remained functional as a phospholipase C (37), an activity that would be required for phagosomal escape and virulence (27). Yet this hypothesis has not been tested, leaving the role of AcpA in Francisella virulence a controversial matter.All studies of Francisella acid phosphatases have been carried out with F. novicida (4, 27, 29, 37), raising the question of significance with regard to the virulent F. tularensis subspecies. In particular, recent whole-genome comparisons between F. novicida and the different Francisella tularensis subspecies have highlighted important intervening sequence (IS)-mediated genome rearrangements in F. tularensis subsp. holarctica and F. tularensis subsp. tularensis strains relative to F. novicida (10). Such rearrangements have disrupted large numbers of open reading frames (ORFs), thereby creating pseudogenes (10) and likely inactivating many functions in virulent F. tularensis strains. For example, Mohapatra et al. (29) have reported that the virulent type A strain Schu S4 is missing a homolog of one of the two hap genes (FTN_0022) present in F. novicida, raising the question of conservation of acid phosphatase-encoding genes in virulent strains. Because phagosomal escape is an essential stage of the Francisella intracellular cycle that is common to F. novicida and F. tularensis, we have postulated that factors required to promote this process must be conserved between these organisms. Here we have compared acid phosphatase-encoding genes in F. novicida and virulent F. tularensis subspecies, and we have generated deletion mutants of the most conserved genes in Schu S4 in order to test their role in the phagosomal escape and pathogenesis of the highly virulent F. tularensis subspecies. We demonstrate that most acid-phosphatase-encoding genes are disrupted in virulent strains and that the most conserved loci are not required for phagosomal escape and virulence.