Bacterial Elements Involved in Catecholamine Mediated Growth Induction

The provision of normally sequestered host Tf or Lf-iron by catecholamines is of considerable significance as it enables pathogenic bacterial species that lack specific binding proteins or uptake systems for Tf or Lf to grow in normally bacterio-static iron-restricted environments, such as blood or serum (Freestone et al. 2008a, b). As a consequence, a considerable amount of data has been elucidated about the bacterial molecular machinery involved in growth responsiveness to the cate-cholamines, and to bacterial-Tf- and Lf-catecholamine interactions. For Gramnegative species such as E. coli (Freestone et al. 2000; Burton et al. 2002; Freestone et al. 2003), Salmonella enterica (Williams et al. 2006) and Bordetella spp. (Anderson and Armstrong 2008) siderophore synthesis and uptake systems have both been shown to be the key elements in the catecholamine-mediated growth induction process. This section will therefore examine the genes involved in cate-cholamine responsiveness for the two most investigated enteric pathogens, E.coli O157:H7 and Salmonella enterica Sv Typhimurium.

For E. coli, the enterobactin synthesis gene entA (Freestone et al. 2000, 2003; Burton et al. 2002) was found to be essential for catecholamine growth induction. Later, work from our laboratory demonstrated that the product of the entF gene was also required, as were also proteins involved in synthesising amino acid precursors for enterobactin, such as AroK and AroD (Freestone, unpublished data). The presence of a functional uptake system for enterobactin was also found to be necessary, as an E.coli tonB mutant failed to grow in the presence of the catecholamine nor-epinephrine (Freestone et al. 2003) or to dopamine, or a variety of dietary catechol compounds (Freestone et al. 2007c). The response of the E.coli siderophore mutants to growth in serum-based medium in the presence of the catecholamine noreipephrine is shown in Fig. 3.4a. In the case of Salmonella, initial unpublished work from our laboratory (Fig. 3.4b) that was later extended by Williams et al. (2006) and Bearson et al. (2008) similarly showed that siderophore synthesis and

1.0E+09

b 1.00E+09

Control

1.00E+08

1.00E+07

1.00E+06

1.00E+04 1.00E+03

Control

Control

Fig. 3.4 The importance of ferrous iron uptake systems in catecholamine-growth induction. Overnight cultures of E. coli O157:H7 (Panel a) and S. enterica (Panel b) were inoculated at approximately 102 CFU/ml into triplicate 1 mL aliquots of serum-SAPI medium (Freestone et al. 2000) containing no additions (Control) 100 (mM norepinephrine (NE) or 100 (mM ferric nitrate (Fe) and incubated statically at 37°C in a 5% CO2 atmosphere for 18 h. The iron was included to demonstrate that failure to grown in the serum-based medium was due to lack of a ferrous iron uptake system, and not sensitivity to the serum itself. Growth of the cultures was enumerated on luria agar as described in Freestone et al. (2000). The results shown are representative data from at least three separate experiments; all data points showed variation of less than 5%

a receptor proteins (fepA, iroN and cirA) were all necessary elements in Salmonella catecholamine growth responsiveness. For Bordetella species, Anderson and Armstrong (2008) have also demonstrated that the presence of an intact enterobac-tin production and uptake system were required for growth-related responses to norepinephrine, such as the ability of the bacteria to acquire iron from Tf in the presence of norepinephrine.

Since we had shown that catecholamine growth induction in serum-based medium involved provision of iron from serum-transferrin (Freestone et al. 2000), we also undertook analyses of the importance of ferric uptake (siderophore) systems in the mechanism by which enteric bacteria uptake iron from Tf and other host iron binding proteins; these investigations are illustrated in Figs. 3.5 and 3.6. Figure 3.5 (our unpublished data) shows the ability of norepinephrine to deliver iron a 140000

S 120000 -

a 100000 -

60000 -

40000

20000

a 140000

S 120000 -

a 100000 -

o ft

60000 -

40000

20000

Indirect

Indirect+

Contact

Contact+

40000 35000 30000 25000 20000 15000 10000 5000 0

Indirect

Indirect+

Contact Contact+

60000 50000 40000 30000 20000 10000 0

Fig. 3.5 The importance of direct contact in E. coli uptake of Tf, Lf and ferritin-complexed iron. Filter-sterilised 55Fe-Tf, 55Fe-Lf and 55Fe-ferritin were prepared as described in Freestone et al. (2000) and added at 2.5x105 cpm/ml, either directly into 5 ml of sterile SAPI medium (Freestone et al. 2000) buffered with 50 mM Tris-HCl, pH 7.5 supplemented with 100 (mM norepinephrine or an equivalent volume of water. Bacteria were incubated either in direct "contact" with the 55Fe-labelled proteins or enclosed within 1-cm diameter dialysis membrane (4 kDa cut-off) ("indirect" contact). An exponential growing culture of wildtype E. coli O157:H7 was added directly to the 55Fe-labelled protein mixtures at 2 x 108 CFU/ml, and incubated at 37°C in a 5% CO2 atmosphere for 4 h, during which time there was essentially no additional growth. The bacteria were then harvested, washed in PBS and assayed for cell numbers and 55Fe incorporation using scintillation counting as described previously (Freestone et al. 2000). a-c: bacterial uptake of 55Fe from 55Fe-Tf (a), 55Fe-Lf (b) and 55Fe-ferritin (c) in the presence and absence of norepinephrine

Indirect

Indirect+

Contact Contact+

Fig. 3.5 The importance of direct contact in E. coli uptake of Tf, Lf and ferritin-complexed iron. Filter-sterilised 55Fe-Tf, 55Fe-Lf and 55Fe-ferritin were prepared as described in Freestone et al. (2000) and added at 2.5x105 cpm/ml, either directly into 5 ml of sterile SAPI medium (Freestone et al. 2000) buffered with 50 mM Tris-HCl, pH 7.5 supplemented with 100 (mM norepinephrine or an equivalent volume of water. Bacteria were incubated either in direct "contact" with the 55Fe-labelled proteins or enclosed within 1-cm diameter dialysis membrane (4 kDa cut-off) ("indirect" contact). An exponential growing culture of wildtype E. coli O157:H7 was added directly to the 55Fe-labelled protein mixtures at 2 x 108 CFU/ml, and incubated at 37°C in a 5% CO2 atmosphere for 4 h, during which time there was essentially no additional growth. The bacteria were then harvested, washed in PBS and assayed for cell numbers and 55Fe incorporation using scintillation counting as described previously (Freestone et al. 2000). a-c: bacterial uptake of 55Fe from 55Fe-Tf (a), 55Fe-Lf (b) and 55Fe-ferritin (c) in the presence and absence of norepinephrine

140000 120000 -100000 -80000 -60000 -40000 20000 0

b 90000

30000

Indirect Indirect+ Contact Contact+

b 90000

30000

Indirect

Indirect+

Contact Contact+

o ft

12000

2000

2000

Fig. 3.6 The importance of ferrous iron uptake systems in Tf iron assimilation. Demonstration of the role of ferrous iron uptake systems in the mechanism of Gram-negative uptake of iron from Tf was carried out using enterobactin siderophore synthesis and uptake mutants (entA and tonB) of E. coli O157:H7 (Freestone et al. 2003) and Salmonella enterica (current study). To test the ability of the strains to acquire iron from 55Fe-Tf, indirect and direct contact assays were performed as described in the legend to Fig. 3.4. a and b, uptake of 55Fe- from 55Fe-Tf by E. coli O157:H7 (a) and S. enterica (b) wildtype, entA and tonB mutants in the presence and absence of 100 (mM norepinephrine or an equivalent volume of water. (c) uptake of 55Fe- from 55Fe-Tf by non-siderophore producing S. epidermidis in the presence and absence of 100 (M norepinephrine or an equivalent volume of water

Indirect Indirect+ Contact Contact+

Fig. 3.6 The importance of ferrous iron uptake systems in Tf iron assimilation. Demonstration of the role of ferrous iron uptake systems in the mechanism of Gram-negative uptake of iron from Tf was carried out using enterobactin siderophore synthesis and uptake mutants (entA and tonB) of E. coli O157:H7 (Freestone et al. 2003) and Salmonella enterica (current study). To test the ability of the strains to acquire iron from 55Fe-Tf, indirect and direct contact assays were performed as described in the legend to Fig. 3.4. a and b, uptake of 55Fe- from 55Fe-Tf by E. coli O157:H7 (a) and S. enterica (b) wildtype, entA and tonB mutants in the presence and absence of 100 (mM norepinephrine or an equivalent volume of water. (c) uptake of 55Fe- from 55Fe-Tf by non-siderophore producing S. epidermidis in the presence and absence of 100 (M norepinephrine or an equivalent volume of water a c

(in the form of 55Fe) from 55Fe-labelled Tf, Lf and ferritin to an enterobactin-producing E. coli O157:H7. In the experiment shown, the bacteria were either partitioned into dialysis tubing ("Indirect" contact), and were therefore only able to acquire 55Fe via secreted iron binding molecules or were in direct contact with the 55Fe-protein ("Contact"). It is clear for Tf, Lf and ferritin that bacteria that had direct contact with the iron binding proteins were able to extract the greatest levels of iron (in the form of 55Fe), particularly if the catecholamine was present. However, even when the iron binding protein was spatially distant from the bacteria, the catecholamine still enabled considerable uptake of the host-protein-complexed 55Fe. In the absence of the stress hormone, the secreted factors released by the bacteria were relatively poor at removing 55Fe from the extracellular Tf and Lf, though effective at scavenging 55Fe from the intracellular ferritin, although in the latter case the presence of norepinephrine still enabled greater E. coli iron assimilation. Figure 3.5 also shows that bacterial uptake of iron from Lf was less than that of Tf. Wally et al. (2006) showed that differences in Tf and Lf iron binding affinity that were due to the variation in the structure of the iron binding domain's inter-lobe linker within the two proteins existed; this region, which is helical in Lf, is unstructured in Tf, making the removal of iron more difficult from Lf, which in contrast to Tf can retain its iron at acidic pH values (Lambert et al. 2005). This could explain the comparative differences in the potency of norepinephrine effects on Tf- and Lf-iron removal observed.

The ability of the catecholamines to complex and directly remove Tf and Lf iron means that bacteria do not have to closely associate with Tf or Lf in order to acquire this iron. Figure 3.5a and b show the importance of the presence of the ferric iron (siderophore) acquisition system in E. coli and Salmonella enterica uptake iron from Tf. Siderophore synthesis and uptake mutants (entA and tonB) along with their wildtype parental strains were incubated in direct contact with 55Fe-Tf, or spatially separated from it within dialysis tubing, and analysed for iron uptake from the proteins (55Fe incorporation) as described in Fig. 3.6. For both E. coli and S. enterica, whether in direct or indirect contact with the 55Fe-Tf, the entA and tonB mutants both showed a significant reduction in the uptake of55Fe when compared with wildtype, indicating the importance of enterobactin synthesis and uptake in the mechanism of catecholamine-mediated host iron acquisition. A similar finding for the role of enterobactin in assimilating catecholamine-mediated Tf iron release by the respiratory pathogen Bordetella bronchiseptica has also been made (Anderson and Armstrong 2008).

An example for the ability of Gram-positive bacteria to access Tf iron in the presence of a catecholamine (norepinephrine) is shown in Fig. 3.5c. The microbe shown is Staphylococcus epidermidis, a bacterium that does not produce sidero-phores. The impact of the lack of a siderophore can be clearly seen, as even when in contact with the 55Fe-Tf, the bacteria were able to acquire only very low levels of 55Fe, which is in marked contrast to the Gram-negative bacterial species already considered. However, in the presence of the catecholamine, the S. epidermidis culture was able to assimilate nearly 30 times more 55Fe. Significantly, we have shown that this iron provision from Tf can lead to massive growth enhancement

(Freestone et al. 1999; Neal et al. 2001 ; Freestone et al. 2008b) as well as the enhancement of biofilm formation in intravenous lines (Lyte et al. 2003) (see also Chap. 8).

0 0

Post a comment