Expression of staphylococcal clumping factor A impedes macrophage phagocytosis

Clumping factor A (ClfA), a fibrinogen-binding protein linked to the Staphylococcus aureus cell wall, is an important virulence factor in infection models, e.g., of septic arthritis. However, the mechanism(s) by which ClfA contributes to the virulence of the bacterium is unknown. In the present study, the impact of ClfA expression on the phagocytosis of S. aureus by macrophages was investigated using clfA-positive and clfA-negative isogenic strains. Furthermore, the possible contribution of ClfA to the proinflammatory and immunostimulatory activity of S. aureus was studied. Our results indicate that ClfA expression significantly protects S. aureus against macrophage phagocytosis. This protection does not require the presence of intact fibrinogen, a ligand for ClfA. ClfA expression by S. aureus enhanced the proliferative response of spleen cells. On the other hand, a clfA mutant strain caused more release of proinflammatory mediators by macrophages than its clfA-positive parental strain. Both the protection against phagocytosis and the enhanced immunostimulatory activity provided by ClfA expression are likely to contribute to the in vivo virulence of S. aureus.     

Attachment, ingestion and intracellular killing of Helicobacter pylori by human peripheral blood mononuclear leukocytes and mouse peritoneal inflammatory macrophages

Abstract The different steps of phagocytosis, attachment, ingestion and intracellular killing of cells of Helicobacter pylori strain 17874 (expressing sialic acid-specific haemagglutinin) and cells of H. pylori strain 17875 (expressing non-sialic acid-specific haemagglutinin) have been studied. More cells of sialopositive H. pylori strain 17874 have been found attached to human peripheral blood mononuclear leukocytes (PBM) and mouse peritoneal inflammatory macrophages (PIM) than cells of sialonegative H. pylori strain 17875. Binding of cells of H. pylori strain 17874 has been significantly inhibited by treatment of phagocytes with neuraminidase. Inhibition of adhesion of these bacteria preincubated with foetuin to normal phagocytic cells has also been found. Well adhering cells of H. pylori strain 17874 were more resistant to killing mechanisms of human PBM and mouse PIM than cells of strain 17875. Good, probably sialic acid-specific haemagglutinin dependent, adhesion of H. pylori bacteria to phagocytes can be considered as an important virulence factor which facilitates the pathogen to avoid the defence mechanisms.     

Opsonin-dependent phagocytosis of bacteria by murine macrophage-like cells

We have investigated the phagocytic properties of the macrophage-like cell line DCH-7, derived from fusion of mouse macrophages with a mouse T-lymphoma cell line. These cells phagocytosed opsonized bacteria. IgG appeared to be the major opsonin forStaphylococcus aureus Wood 46 as well as for threeEscherichia coli strains; complement components were not required as opsonins. Intracellular bacteria survived to a large extent. This model system should be a useful tool for studying the process of phagocytosis and phagocytic killing of bacteria.     

Correlation of in vitro properties of Rhodococcus (Corynebacterium) equi with virulence for mice

To study the virulence of Rhodococcus (Corynebacterium) equi, seven ATCC strains of different serotypes were tested for their LD50 in mice, clearance of the organism from the lungs and spleen following intravenous or intratracheal inoculation, and in vitro interaction with murine peritoneal macrophages. Strains ATCC 33704 and 33705 were virulent for mice and multiplied in the lungs and spleen, resulting in death of the animal in 5 days. The other five strains were avirulent for mice. The number of bacteria in the lungs and spleen of mice given these five strains decreased immediately. Pulmonary clearance of strains ATCC 33703, 33706, and 33707 was significantly more rapid than that of the virulent strains ATCC 33704 and 33705 12 hr after inoculation. Complete clearance of the avirulent strain ATCC 33707 occurred by day 14, while that of virulent ATCC 33704 and 33705 strains occurred by day 30. The virulent strains ATCC 33704 and 33705 were resistant not only to phagocytosis but also to intracellular killing by macrophages. Strains ATCC 33702 and 33706 were rapidly killed by macrophages although they were rather resistant to phagocytosis. Strain ATCC 33703 was easily phagocytized though resistant to killing by macrophages. The most avirulent strains, ATCC 33707 and 6939, were easily phagocytized and rapidly killed by macrophages. These results indicate that virulence appeared to be related to the ability of the organisms to resist clearance from the lungs and spleen and to resist phagocytosis and intracellular killing by macrophages.     

A bacterial regulatory RNA attenuates virulence,spread and human host cell phagocytosis

Staphylococcus aureus pathogenesis is directed by regulatory proteins and RNAs. We report the case of an RNA attenuating virulence and host uptake, possibly to sustain commensalism. A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model. S. aureus deleted for sprC became more virulent and increased bacterial dissemination in colonized animals. Conversely, inducing SprC expression lowered virulence and the bacterial load. Without sprC, S. aureus phagocytosis by monocytes and macrophages was higher, whereas bacteria were internalized at lower yields when SprC expression was stimulated. Without sprC, higher internalization led to a greater number of extracellular bacteria, facilitating colonization. SprC expression decreased after phagocytosis, concurring with the facilitated growth of bacteria lacking the sRNA in the presence of an oxidant. The major staphylococcal autolysin facilitates S. aureus uptake by human phagocytes. ATL proved to be negatively regulated by SprC. The SprC domains involved in pairing with atl mRNA were analyzed. The addition of ATL reduced phagocytosis of bacteria lacking sprC with no effects on wild-type bacterial uptake, implying that SprC influences phagocytosis, at least in part, by controlling ATL. Since the control of SprC on ATL was modest, other factors must contribute to atl regulation.     

Icm/Dot-dependent upregulation of phagocytosis by Legionella pneumophila

Legionella pneumophila is the causative agent of Legionnaires' disease, a severe pneumonia. Dependent on the icm/dot loci, L. pneumophila survives and replicates in macrophages and amoebae within a specialized phagosome that does not fuse with lysosomes. Here, we report that phagocytosis of wild-type L. pneumophila is more efficient than uptake of icm/dot mutants. Compared with the wild-type strain JR32, about 10 times fewer icm/dot mutant bacteria were recovered from HL-60 macrophages in a gentamicin protection assay. The defect in phagocytosis of the mutants could be complemented by supplying the corresponding genes on a plasmid. Using fluorescence microscopy and green fluorescent protein (GFP)-expressing strains, 10-20 times fewer icm/dot mutant bacteria were found to be internalized by HL-60 cells and human monocyte-derived macrophages (HMMPhi). Compared with icm/dot mutants, wild-type L. pneumophila infected two to three times more macrophages and yielded a population of highly infected host cells (15-70 bacteria per macrophage) that was not observed with icm/dot mutant strains. Wild-type and icmT mutant bacteria were found to adhere similarly and compete for binding to HMMPhi. In addition, wild-type L. pneumophila was also phagocytosed more efficiently by Acanthamoeba castellanii, indicating that the process is independent of adherence receptor(s). Wild-type L. pneumophila enhanced phagocytosis of an icmT mutant strain in a synchronous co-infection, suggesting that increased phagocytosis results from (a) secreted effector(s) acting in trans.     

Expression and distribution of very late antigen-5 in mouse peritoneal macrophages upon ingestion of fibronectin-bound Staphylococcus aureus

Many pathogens colonize host tissues by binding to the extracellular matrix via their cell surface adhesion molecules, which are called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). Staphylococcus aureus expresses several of these adhesion molecules, some of which bind to fibronectin. Of these adhesion molecules, fibronectin-binding proteins play a role in the pathogenicity of S. aureus, although it is not yet clear whether they enhance its virulence. We have previously shown that fibronectin-bound S. aureus is efficiently phagocytosed by thioglycolate-induced mouse peritoneal macrophages. Bacterial ingestion is mediated by Very Late Antigen-5 (VLA-5; alpha5beta1 integrin) and is accompanied by the formation of adhesion complexes. Here we show that the expression of VLA-5 is restricted to thioglycolate-induced inflammatory macrophages and is not found in the resident macrophages. When cells were in suspension, alpha5 integrin was not expressed on the surface of either resident or inflammatory macrophages, whereas in adherent cells, this integrin was distributed on the surface of inflammatory but not resident macrophages. A high level of this integrin was present in the cytoplasmic region only in inflammatory macrophages. In agreement with this, fibronectin-mediated phagocytosis of S. aureus was observed only in the inflammatory macrophages. In inflammatory macrophages ingesting fibronectin-bound S. aureus, alpha5 integrin was concentrated close to the phagocytosed bacteria. This change in distribution was not found in macrophages ingesting untreated bacteria. Together with our previous work, these results indicate that, upon ingestion of fibronectin-bound S. aureus, VLA-5 accumulates in the area of phagocytosis in inflammatory macrophages, where it forms adhesion complexes.     

Low-level iron-dependent mutants of Listeria monocytogenes and their virulence in macrophages

Listeria monocytogenes is an opportunistic intracellular pathogen capable of growth that requires iron for growth within phagocytic cells and virulence expression. In the presence of an appropriate concentration tropolone, an iron-chelating agent, growth of L. monocytogenes is completely inhibited. However, this inhibition can be relieved by addition of dopamine, norepinephrine, or ferric citrate. By selection on streptonigrin medium supplemented with tropolone and norepinephrine, we have obtained two spontaneous mutants, Lm-8 and Lm-15, with the same iron dependence but lower iron dependence than the wild-type Lm-B38. The association between iron requirement and virulence of the two mutants and the wild type was studied in the J774 macrophage cell line. One hour after phagocytosis by the J774 macrophage cell line, the two mutants and the parental strain displayed no difference in the number of phagocytosed bacteria. Twenty-four hours after phagocytosis, the number of bacteria within the surviving macrophages was identical for the wild strain and the two clones. However, only 40% of macrophage cells infected with Lm-8 and 90% of those infected with Lm-15 were alive after 24 h in comparison with macrophage cells infected with the parental strain Lm-B38. These data demonstrate that there is no direct correlation between iron requirement and virulence of L. monocytogenes in the J774 macrophage cell line.     

Early interactions of Flavobacterium psychrophilum with macrophages of rainbow trout Oncorhynchus mykiss

The early interactions of a low and a highly virulent Flavobacterium psychrophilum strain with head kidney and spleen macrophages of rainbow trout Oncorhynchus mykiss were characterized. The highly virulent strain was killed 5.8 to 11 times less frequently than the low virulent strain. The head kidney macrophages showed a microbicidal activity approximately twice as high as that of the spleen macrophages. A 2- to 3-fold higher production of reactive oxygen species (ROS) was induced by the highly virulent strain than by the low virulent one. The head kidney macrophages produced approximately twice as much ROS as the spleen macrophages. The low virulent strain was killed approximately 10 times more frequently by H2O2 than was the highly virulent strain. In spleen macrophages, the highly virulent strain caused twice as much cytotoxic effects compared to the low virulent strain. In conclusion, virulence in F. psychrophilum appears to be correlated with higher O. mykiss macrophage cytotoxicity and resistance to ROS and, therefore, with enhanced resistance to bacterial killing. Moreover, due to lower ROS production, spleen macrophages have a lower antimicrobial action against F. psychrophilum, compared to head kidney macrophages and, thus, might form a 'safe site' in which bacteria can reside.     

Effect of insulin and isoproterenol on macrophage phagocytic activity

To reveal the influence of cyclic adenosine monophosphate (cAMP) on the completion of the phagocytosis of salmonellae, the influence of insulin and isoproterenol on the phagocytic activity of peritoneal macrophages obtained from mice infected with S. typhimurium strains differing in virulence was studied in vitro. The study showed that isoproterenol, while increasing the intracellular content of cAMP, suppressed the bactericidal properties of macrophages with respect to salmonellae, whereas insulin decreased the level of cAMP in the cells and thus facilitated more rapid and complete digestion of ingested bacteria irrespective of their virulence.     

In vitro interactions between bacteria, osteoblast-like cells and macrophages in the pathogenesis of biomaterial-associated infections

Biomaterial-associated infections constitute a major clinical problem that is difficult to treat and often necessitates implant replacement. Pathogens can be introduced on an implant surface during surgery and compete with host cells attempting to integrate the implant. The fate of a biomaterial implant depends on the outcome of this race for the surface. Here we studied the competition between different bacterial strains and human U2OS osteoblast-like cells (ATCC HTB-94) for a poly(methylmethacrylate) surface in the absence or presence of macrophages in vitro using a peri-operative contamination model. Bacteria were seeded on the surface at a shear rate of 11 1/s prior to adhesion of U2OS cells and macrophages. Next, bacteria, U2OS cells and macrophages were allowed to grow simultaneously under low shear conditions (0.14 1/s). The outcome of the competition between bacteria and U2OS cells for the surface critically depended on bacterial virulence. In absence of macrophages, highly virulent Staphylococcus aureus or Pseudomonas aeruginosa stimulated U2OS cell death within 18 h of simultaneous growth on a surface. Moreover, these strains also caused cell death despite phagocytosis of adhering bacteria in presence of murine macrophages. Thus U2OS cells are bound to loose the race for a biomaterial surface against S. aureus or P. aeruginosa, even in presence of macrophages. In contrast, low-virulent Staphylococcus epidermidis did not cause U2OS cell death even after 48 h, regardless of the absence or presence of macrophages. Clinically, S. aureus and P. aeruginosa are known to yield acute and severe biomaterial-associated infections in contrast to S. epidermidis, mostly known to cause more low-grade infection. Thus it can be concluded that the model described possesses features concurring with clinical observations and therewith has potential for further studies on the simultaneous competition for an implant surface between tissue cells and pathogenic bacteria in presence of immune system components.     

The enhancing effect of cell-wall aggressin on the virulence of Staphylococcus aureus in normal and irradiated mice and in man

Mice irradiated with lethal doses of total body X-irradiation are very susceptible to Staphylococcus aureus. A difference in virulence between a “mouse-virulent” strain and a “mouse-non-virulent” strain which lacks cell-wall aggressin could nevertheless be demonstrated. The virulence of the “mouse-non-virulent” strain could be increased by adding a cell-wall preparation with aggressin-activity (DOCR) from a “mouse-virulent” strain to the inoculum. When injected together with a dose of bacteria lower than the minimum pus-forming dose a lesion-enhancing effect of DOCR from a “mouse-virulent” strain was also demonstrated in man.     

Changes in the cAMP level in macrophages during the phagocytosis of Salmonella typhimurium. II. Effect of the virulence of microorganisms and the immune status of the macrophages on the dynamics of changes in cAMP levels

The dynamics of changes in the level of intracellular cyclic AMP in nonimmune and immune macrophages in the process of the phagocytosis of S. typhimurium virulent strain, its heated variant and its mutant with low virulence was studied. A transient increase in the level of cyclic AMP was shown to occur during the first 30 minutes of phagocytosis; this increase did not depend on the kind of the phagocytized object and on the activity of macrophages and neither had it any influence on the outcome of phagocytosis. The virulent strain also induced the three-fold increase of the level of cyclic AMP in nonimmune macrophages prior to their disintegration caused by the cytopathogenic action of the microorganism. In immune macrophages the virulent strain did not induce the secondary increase of the level of cyclic AMP.     

Comparison of the responses of peritoneal macrophages from Japanese flounder (Paralichthys olivaceus) against high virulent and low virulent strains of Edwardsiella tarda

In vivo infection studies in Japanese flounder (Paralichthys olivaceus) demonstrated that the number of viable cells of the virulent strain (NUF251) of Edwardsiella tarda increased gradually in kidney and hepato-pancreas after intraperitoneal injection, but the low virulent strain (NUF194) did not. To gain insight into the virulence factors of E. tarda, in vitro responses of Japanese flounder (P. olivaceus) peritoneal macrophages to these strains were compared in terms of phagocytosis, bactericidal activity, and reactive oxygen species (ROS) generation as measured by chemiluminescence (CL) responses. Microscopic observation revealed that these two strains of E. tarda were phagocytosed by the peritoneal macrophages, and there was no significant difference in the mean numbers of ingested bacteria per macrophage between these strains. A gradual increase in the number of viable cells of the highly virulent strain within macrophages was observed during 9h post-phagocytosis, whereas no significant replication of the low virulent strain within macrophages was detected. These results suggest that the virulent strain of E. tarda has an ability to survive and replicate within macrophages, while the low virulent strain has no such ability. When the peritoneal macrophages were exposed to the opsonized low virulent E. tarda strain, a rapid increase in CL response was induced. However, the highly virulent strain caused only background level of CL response. By the subsequent stimulation with phorbol myristate acetate, the macrophages exposed to the virulent E. tarda strain showed extremely higher CL response than that of the one exposed to the low virulent E. tarda strain. These results suggest that the virulent E. tarda prevents the activation of ROS generation system during phagocytosis, though the system is still capable of responding to other stimulation. The virulent strain significantly reduced the CL response induced by xanthine/xanthine oxidase system, while the low virulent strain had almost no effect. Furthermore, the virulent strain showed greater resistance to H(2)O(2) than the low virulent strain. Our results suggest that the virulent strain of E. tarda is highly resistant to ROS, and such ability might allow the organism to survive and multiply within phagocytes, and may serve to disseminate E. tarda throughout the host during in vivo infection.     

Mouse Virulence of K(L) Antigen-Containing Strains of Escherichia coli

The mouse virulence of two K antigen-containing (L variety) strains of Escherichia coli (serotype O2:K1) isolated from human septicemia, and of their variants which lacked K antigen, was studied. The strains containing envelope antigen (K+) were highly virulent when injected intracerebrally or when suspended in mucin and injected intraperitoneally. After intraperitoneal injection of E-107 K+ (but not K−), there was a marked initial growth in the peritoneal cavity followed by bacteremia and infection of all the organs examined. In the mucin-enhanced lethal infection, this growth continued until death of the animal; in the nonlethal infection, growth ceased and the count dropped quickly after approximately 5 hr. Host defenses were depressed greatly by intraperitoneally, but not intravenously, administered mucin. Bacteria were most virulent when injected intraperitoneally. In vitro phagocytosis of the K+ bacteria required opsonins not needed for phagocytosis of the smooth K− variants. Opsonins were found in immunized rabbit and normal mouse sera. Immune rabbit sera contained antibodies with anti-K specificity which were opsonic in vitro and highly protective in vivo when administered passively. There appears to be a lesser anti-O opsonic and protective activity involving one of the strains (E-107 K+), and colonial morphology, agglutination, and absorption tests indicated a low amount of K antigen on this organism. No anti-O opsonic or protective activity could be shown involving the other strain (E-102 K+). When standard serological typing procedures were used, these two strains appeared to be identical serologically, but they differed greatly in sensitivity to immune rabbit serum in phagocytosis experiments in vitro.     

Comparison of invasion of fibroblasts and macrophages by high- and low-virulence Leptospira strains: colonization of the host-cell nucleus and induction of necrosis by the virulent strain

The infection cycle of low- and high-virulence strains of Leptospira interrogans was compared in fibroblasts and macrophages. L. interrogans serovar Lai strain Lai was used as a representative high-virulence strain, while L. interrogans serovars Pomona strain Luo was used as a low-virulence strain. L. biflexa serovar Patoc strain Patoc I, a nonparasitic strain of Leptospira, was used as a control. Both the high- and low-virulence strains could adhere to fibroblasts and macrophages using one or both ends of the spirochete, which was followed by phagocytosis of both strains. Both strains adhered more strongly to macrophages than fibroblasts. However, the high-virulence strain could invade the host-cell nucleus, while the low-virulence strain remained in phagosomes. The L. biflexa strain neither adhered to nor invaded either cell type. Both of the L. interrogans strains also induced cell death (mostly necrosis) of macrophages, whether or not the spirochetes were viable, suggesting that leptospiral virulence is unrelated to macrophage death. However, the high-virulence strain induced mainly necrosis in fibroblasts, while the low-virulence strain induced more apoptosis. Thus, the main feature distinguishing the two L. interrogans strains is the ability of the high-virulence strain to invade the host-cell nucleus and induce pro-inflammatory necrosis in fibroblasts.     

Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death,and bacterial escape and dissemination

The success of Staphylococcus aureus as a pathogen is partly attributable to its ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of phagocytes. Here, we have studied the interaction of methicillin‐resistant S. aureus (MRSA) strain USA300 with murine RAW 264.7 and primary human macrophages using molecular imaging and single cell analysis to obtain an unprecedented understanding of the interaction between the macrophage and MRSA. Herein we demonstrate that macrophages fail to control intracellular infection by MRSA USA300 despite trafficking the bacteria into mature phagolysosomes. Using fluorescence‐based proliferation assays we also show that intracellular staphylococci proliferate and that replication commences while the bacteria are residing in mature phagolysosomes hours after initial phagocytosis. Finally, live‐cell fluorescence video microscopy allowed for unprecedented visual insight into the escape of MRSA from macrophages, demonstrating that the macrophages die through a pathway characterized by membrane blebbing and activation of caspase‐3 followed by acquisition of the vital dye propidium iodide. Moreover, cell death precedes the emergence of MRSA from infected macrophages, and these events can be ablated by prolonged exposure of infected phagocytes to gentamicin.     

Stimulation and inhibition of polymorphonuclear leukocytes phagocytosis by lipoamino acids isolated from Serratia marcescens

Abstract The role of the lipoamino acids (serratamolide and ornithine lipid), membrane lipid components of Serratia marcescens , was examined in phagocytosis and phagosome-lysosome fusion of human peripheral polymorphonuclear leukocytes. A mutant strain of Serratia marcescens (NS 38-09) lacking serratamolide was actively phagocytosed by human PMN, while the wild-type strain (NS 38) producing serratamolide was more resistant to phagocytosis by human PMN. Phagocytosis of killed Staphylococcus aureus coated with lipoamino acid (serratamolide), showed that they were more resistant to phagocytosis by PMN, while the cells coated with ornithine lipid or serratamic acid were phagocytosed more actively. Staphylococci coated with phosphatidylethanolamine or phosphatidylglycerol had no significant effect on phagocytosis by PMN. Phagosome-lysosome fusion by PMN labelled with acridine orange was examined by fluorescence microscopy. The fusion indices of lipoamino acid-coated staphylococci were the same as that of controls. Further, ornithine lipid-coated staphylococci stimulated the release of superoxide anion from PMN slightly, but serratamolide did not. These results suggested that serratamolide may contribute to the virulence of S. marcescens in vitro.     

Lung surfactant protein A (SP-A) enhances serum-independent phagocytosis of bacteria by alveolar macrophages.

Surfactant protein A (SP-A) is the main protein component of lung surfactant. We studied the involvement of SP-A in body defense, i.e., effect of SP-A on the phagocytosis of bacteria by alveolar macrophages. We show here that SP-A enhances the phagocytosis of some non-opsonized bacteria: Escherichia coli growing logarithmically (E. coli/log), Pseudomonas aeruginosa/log as well as from stationary phase (P. aeruginosa/stat) and Staphylococcus aureus/log. Furthermore, not only serum-independent phagocytosis was effected by SP-A but also phagocytosis of serum-opsonized S. aureus/stat. No effect of SP-A on phagocytosis was observed with E. coli/stat neither on serum-independent nor on serum-dependent phagocytosis and on phagocytosis of non-opsonized S. aureus/stat. Thus, effect of SP-A on phagocytosis is dependent on bacterial species and on the growth phase of the microorganisms, and this effect is concentration dependent. We studied two different human recombinant SP-As and SP-A isolated from lung lavage material from proteinosis patients. These SP-A molecules contain different isomeric chains, and they differ in complexity of their structure. Qualitatively, we found the same effect with all three substances. Quantitatively, the proteinosis SP-A that forms the most complex structure was the most effective. Taken together, we demonstrated a stimulating effect of SP-A on serum-independent as well as on serum-dependent phagocytosis of bacteria by alveolar macrophages, both depending on species and growth phase of the bacteria.